JP6946486B2 - Nucleic acid containing or encoding a histone stem-loop and a poly (A) sequence or polyadenylation signal to increase the expression of the encoded tumor antigen. - Google Patents

Description

The present invention comprises a coding region encoding at least one peptide or protein containing a tumor antigen or fragment, variant, or derivative thereof, at least one histone stem loop, and a poly (A) sequence or polyadenylation signal. Containing or encoding a nucleic acid sequence. In addition, the invention provides the use of the nucleic acid to increase the expression of the encoded peptide or protein. It also discloses, for example, the use of pharmaceutical compositions for use in the treatment of cancer or tumor diseases, particularly the nucleic acids for preparing vaccines. The present invention further uses nucleic acids containing or encoding histone stem-loops and poly (A) sequences or polyadenylation signals to express peptides or proteins containing tumor antigens or fragments, variants, or derivatives thereof. The method of increasing is described.

Aside from cardiovascular and infectious diseases, tumor and cancer diseases are one of the most common causes of death in modern society and are often associated with considerable costs in treatment and subsequent rehabilitation procedures. .. Treatment of tumors and cancer diseases largely depends, for example, on the type and age of the tumor that has developed, the distribution of cancer cells in the patient being treated, and the like. Currently, cancer treatment is conventional with the use of radiation therapy or chemotherapy in addition to invasive surgery. However, such conventional treatments typically place so much stress on the immune system that in some cases they can only be used in a limited way. Moreover, most of these conventional treatments require a long rest period between individual treatments to regenerate the immune system.

Therefore, in recent years, supplementary strategies have been studied in addition to such "conventional treatments" to eliminate or at least reduce the effects of such treatments on the immune system. One such supplemental therapy, specifically, includes a gene therapy approach or a gene vaccine, which have already been found to be very promising in the treatment or support of such conventional therapies. ..

Gene therapy and gene vaccines are molecular medical methods that have already been shown to be effective in the treatment and prevention of diseases, and generally show significant effects in daily medical practice, especially in the treatment of the above diseases. Both gene therapy and gene vaccines introduce a nucleic acid into a patient's cell or tissue and then process the information encoded by the nucleic acid introduced into the cell or tissue, i.e. the desired polypeptide (ie). It is based on expressing (protein).

In gene therapy approaches, DNA is typically used, even though RNA is also known in recent developments. Importantly, in all these gene therapy approaches, mRNA acts as a messenger of sequence information for the encoded protein, regardless of whether DNA, viral RNA, or mRNA is used.

RNA is generally considered to be an unstable molecule. This is because it is well known that RNase is ubiquitous and difficult to inactivate. In addition, RNA is also chemically more unstable than DNA. Therefore, it is perhaps surprising that the "default state" of mRNA in eukaryotic cells is characterized by relatively stable and requires a specific signal to promote the degradation of individual mRNAs. be. The main reason for this finding is believed to be that intracellular mRNA degradation is mostly catalyzed by exonucleases. However, eukaryotic mRNA terminals are protected from these enzymes by specific terminal structures and related proteins (5'end m7GpppN, and typically 3'end poly (A) sequences). .. Therefore, the removal of these two terminal modifications is considered to be the rate-determining factor for mRNA degradation. Stabilizing elements have been characterized in the 3'UTR of alpha-globin mRNA, but RNA sequences that affect eukaryotic mRNA turnover are typically by promoting deadenylation. It acts as a promoter for decomposition (outlined in Non-Patent Document 1).

As mentioned above, the 5'end of eukaryotic mRNA is typically post-transcriptionally modified to have a methylated CAP structure, such as m7GpppN. Apart from its role in RNA splicing, stabilization, and transport, the CAP structure significantly enhances the recruitment of the 40S ribosomal subunit to the 5'end of the mRNA at the onset of translation. The latter function requires recognition of the CAP structure by the eukaryotic translation initiation factor complex eIF4F. The poly (A) sequence further stimulates translation through increased recruitment of the 40S subunit into mRNA, an effect that requires the intervention of a poly (A) binding protein (PABP). PABP has recently been demonstrated to physically interact with eIF4G, which is part of the CAP-bound eIF4F complex. Therefore, a closed-loop model of translation initiation in capped polyadenylated mRNA was claimed (Non-Patent Document 2).

Almost all eukaryotic mRNAs are terminated with a poly (A) sequence added to their 3'end by a ubiquitous cleavage / polyadenylation mechanism. The presence of the poly (A) sequence at the 3'end is one of the most distinctive features of eukaryotic mRNA. After cleavage, most premRNAs acquire a polyadenylated tail, with the exception of replication-dependent histone transcripts. In this regard, 3'end processing is a nuclear co-transcriptional process that promotes the transport of mRNA from the nucleus to the cytoplasm and affects the stability and translation of mRNA. The formation of the 3'end is a two-step reaction directed by a cleavage / polyadenylation mechanism, with two sequence elements in the pre-mRNA (pre-mRNA) (six highly conserved nucleotides AAUAAA (polyadenylation). It depends on the presence of the signal) and the downstream G / U rich sequence). In the first step, premRNA is cleaved between these two elements. In the second step, which is closely related to the first step, the newly formed 3'end is extended by the addition of a poly (A) sequence consisting of 200 to 250 adenirates, which later It affects all aspects of mRNA metabolism, including mRNA export, stability, and translation (Non-Patent Document 3).

The only known exception to this rule is replication-dependent histone mRNAs that terminate at histone stemloops instead of poly (A) sequences. An example of a histone stem-loop sequence is described by Lopez et al. (Non-Patent Document 4).

Stem-loops in histone pre-mRNA typically follow the purin-rich sequence known as the downstream histone element (HDE). These premRNAs are processed in the nucleus by single nucleotide strand breaks about 5 nucleotides downstream of the stem loop, catalyzed by U7 snRNP through base pairing of U7 snRNA with HDE. The 3'-UTR sequence containing the histone stem-loop structure and the downstream histone element (HDE) (the binding site of U7 snRNP) is commonly referred to as the histone 3'-processing signal (see, eg, Non-Patent Document 5).

Histone synthesis is regulated in response to the cell cycle because the newly synthesized DNA needs to be packaged in chromatin. Increased synthesis of histone proteins in S phase is achieved by transcriptional activation of histone genes and post-transcriptional regulation of histone mRNA levels. Histone stem-loops have been shown to be essential for all post-transcriptional processes of histone expression regulation. It is required for efficient processing, export of mRNA to the cytoplasm, loading into polyribosomes, and regulation of mRNA stability.

In connection with the above, a 32 kDa protein that binds to the histone stem-loop at the 3'end of the histone message in both the nucleus and cytoplasm has been identified. The expression level of this stem-loop binding protein (SLBP) is regulated by the cell cycle and is highest in S phase when histone mRNA levels increase. SLBP is required for efficient 3'end processing of histon premRNA by U7 snRNP. After completion of processing, SLBP is still bound to the stem-loop at the end of the mature histone mRNA and stimulates the translation of the mature histone mRNA into histone protein in the cytoplasm (Non-Patent Document 3). Interestingly, the RNA-binding domain of SLBP is conserved across metazoans and protozoa (Non-Patent Document 4), and binding to histone stem-loop sequences depends on stem-loop structure, as well as minimal. The binding site has been shown to contain at least 3 nucleotides in 5'and 2 nucleotides in 3'of the stem-loop (Non-Patent Documents 6 and 7).

Histone genes are generally classified as "replication-dependent" histones in which the mRNA terminates in the histone stemloop or "replacement" histones in which mRNA with a poly (A) tail instead results. In rare cases, mRNAs containing histone stem-loops and poly (A) or oligo (A) in their 3'have been identified in nature. Using luciferase as a reporter protein, Sanchez et al. Studyed the effect of a naturally occurring oligo (A) tail added to the histone stem-loop 3'of histone mRNA during egg formation in African Tsumegaeru, said oligo. (A) We found that the tail is the active part of the translational repression mechanism that silences histone mRNA during egg formation, and that its removal is part of the mechanism that activates the translation of histone mRNA (non-patentable). Document 8).

In addition, taking advantage of the intron-containing presence of the globin gene as opposed to the intron-free histone gene, the marker protein alphaglobin for replication-dependent histone regulation requirements and mRNA stability at the level of premRNA processing. Was investigated using an artificial construct encoding. For this purpose, constructs were created in which the alpha globin coding sequence is followed by a histone stem-loop signal (histone stem-loop followed by a histone downstream element) and a polyadenylation signal (Non-Patent Documents 9-11).

In another approach, Luescher et al. Studyed the cell cycle-dependent regulation of recombinant histone H4 genes. A construct was prepared in which the H4 coding sequence was followed by a histone stem-loop signal and a polyadenylation signal, and the two processing signals were accidentally separated by the galactokinase coding sequence (Non-Patent Document 12).

In addition, Stauber et al. Identified the smallest sequence required to regulate histone H4 mRNA levels in a cell cycle-dependent manner. In these studies, a construct containing the coding sequence of the selection marker xanthine: guanine phosphoribosyl transferase (GPT) was used before the histone stem-loop and the subsequent polyadenylation signal (Non-Patent Document 13).

In the study of histone pre-mRNA processing, Wagner et al. Used a reporter construct in which EGFP was placed between the histone stem-loop signal and the polyadenylation signal so that EGFP was expressed only when histone pre-mRNA processing was interrupted. Factors required for cleavage of histone premRNA were identified (Non-Patent Document 14).

It should be noted that translation of polyadenylated mRNA usually requires the 3'poly (A) sequence to be close to the 5'CAP. It is mediated through a protein-protein interaction between the poly (A) binding protein and the eukaryotic initiation factor eIF4G. A similar mechanism has been elucidated for replication-dependent histone mRNA. In this regard, Gallie et al. Functionally have histone stem-loops on poly (A) sequences in that they increase translation efficiency to establish efficient translation levels and are co-dependent on 5'-CAP. It shows that they are similar. Gallie et al. Found that histone stem-loops are necessary and sufficient to increase the translation of reporter mRNA in infected Chinese hamster ovary cells, but must be located at the 3'end for optimal functioning. Indicated. Therefore, as with the poly (A) tail in other mRNAs, the 3'end of these histone mRNAs is considered essential for translation in vivo and functionally similar to the poly (A) tail (non-). Patent Document 15).

Furthermore, SLBP has been shown to bind to cytoplasmic histone mRNA and be required for its translation. SLBP does not interact directly with eIF4G, but the domain required for histone mRNA translation interacts with the recently identified protein SLIP1. In a further step, SLIP1 interacts with eIF4G to cyclize histone mRNA and support efficient translation of histone mRNA by a mechanism similar to the translation of polyadenylated mRNA.

As mentioned above, gene therapy approaches typically use DNA to transport coding information to cells, which in turn then naturally to ensure that the encoded therapeutic or antigenic protein is expressed. It is transcribed into an element of the mRNA present, particularly an mRNA having a 5'-CAP structure and a 3'poly (A) sequence.

However, in many cases, expression systems based on the introduction of nucleic acids across a patient's cells or tissues, followed by the expression of the desired polypeptide encoded by these nucleic acids, are either DNA or RNA. It does not indicate the desired or even required expression levels that can enable efficient treatment, whether or not.

In related arts, various attempts have been made to increase the expression level of the encoded protein in vitro and / or in vivo by using a particularly improved expression system. The methods commonly described in the art for increasing expression have traditionally been based on the use of expression vectors or cassettes containing specific promoters and corresponding regulatory elements. Since these expression vectors or cassettes are typically limited to a particular cell line, these expression systems must be adapted for use in a variety of cell lines. The expression vector or cassette thus adapted is then usually transfected into cells and typically processed according to the particular cell line. Therefore, nucleic acid molecules capable of expressing proteins encoded by cell-specific systems in target cells, independent of promoters and regulatory elements specific for a particular cell type, are predominantly preferred. In this regard, it is possible to distinguish between the mRNA stabilizing element and the element that enhances the translation efficiency of mRNA.

An mRNA having an optimized coding sequence and generally suitable for such a purpose is described in Patent Document 1. For example, Patent Document 1 describes mRNA that is stabilized in general form and whose coding region is optimized for translation. Patent Document 1 also discloses a method for determining sequence modification. Patent Document 1 also describes the possibility of substituting adenyl and uracil nucleotides in the mRNA sequence to increase the guanine / cytosine (G / C) content of the sequence. According to Patent Document 1, such substitutions and indications for increasing G / C content can be used not only for gene therapy applications, but also for gene vaccines in the treatment of cancer or infectious diseases. .. In this regard, Patent Document 1 generally refers to a sequence as a basic sequence for making such modifications, wherein the modified mRNA encodes at least one bioactive peptide or polypeptide. Translated, for example, in patients who have not received any treatment, have received inappropriate treatment, or have received incorrect treatment. Alternatively, Patent Document 1 proposes an antigen, for example, an mRNA encoding a tumor antigen or a viral antigen, as a basic sequence for making such a modification.

In a further approach to increasing the expression of the encoded protein, Patent Document 2 describes long poly (A) sequences for mRNA stability and translational activity (specifically, longer than 120 bp) and beta globin genes. It describes the beneficial effects with a combination of at least two 3'untranslated regions.

However, despite all the documentation of these latter related technologies already attempting to provide highly efficient tools for gene therapy approaches, as well as improved mRNA stability and translational activity. However, there remains the problem that the use of RNA is generally less stable than DNA vaccines and gene therapy approaches using DNA. Thus, preferably for gene therapy and gene therapy, a gene therapy approach that can better provide the encoded protein in vivo, for example, through further improved mRNA stability and / or translational activity. And there is still a need in the art to provide improved tools for gene therapy or as an adjunct therapy to conventional therapies as described above.

Moreover, despite all advances in the art, efficient expression (recombinant expression) of peptides or proteins encoded in cell-free systems, cells, or organisms remains a challenging task.

International Publication No. 02/098443 Pamphlet (CureVac GmbH) International Publication No. 2007/036366 Pamphlet

Meyer, S.M. , C.I. Temme, et al. (2004), Crit Rev Biochem Mol Biol 39 (4): 197-216. Michel, Y. et al. M. , D. Pontet, et al. (2000), J Biol Chem 275 (41): 32268-76. Dominski, Z. and W. F. Marzluff (2007), Gene 396 (2): 373-90. Davila Lopez, M. et al. , & Samuelsson, T.M. (2008), RNA (New York, NY), 14 (1), 1-10. doi: 10.261 / rna. 782308. Chodchoy, N.M. , N. B. Pandy, et al. (1991). Mol Cell Biol 11 (1): 497-509. Pandy, N. et al. B. , Et al. (1994), Molecular and Cellular Biology, 14 (3), 1709-1720. Williams, A.M. S. , & Marzflff, W.M. F. , (1995), Nucleic Acids Research, 23 (4), 654-662. Sanchez, R.M. and W. F. Marzflff (2004), Mol Cell Biol 24 (6): 2513-25 Whitenow, E.I. , Et al. (1986). Nucleic Acids Research, 14 (17), 7059-7070. Pandy, N. et al. B. , & Marzflff, W.M. F. (1987). Molecular and Cellular Biology, 7 (12), 4557-4559. Pandy, N. et al. B. , Et al. (1990). Nucleic Acids Research, 18 (11), 3161-3170 Luescher, B. et al. et al. , (1985). Proc. Natl. Acad. Sci. USA, 82 (13), 4389-4393 Stauber, C.I. et al. , (1986). EMBO J, 5 (12), 3297-3303 Wagner, E.I. J. et al. , (2007). Mol Cell 28 (4), 692-9 Gallie, D.M. R. , Lewis, N. et al. J. , & Marzflff, W.M. F. (1996), Nucleic Acids Research, 24 (10), 1954-1962.

Therefore, the underlying object of the present invention is preferably the further of mRNA in relation to nucleic acids known in related techniques for use in gene vaccines in the therapeutic or prophylactic treatment of cancer or tumor disease. To provide additional and / or alternative methods for increasing the expression of the encoded protein through stabilization and / or increased translation efficiency of the mRNA.

The object is solved by the subject of the invention in the appended claims. Specifically, the underlying object of the present invention is according to the first aspect.
a) A coding region encoding at least one peptide or protein containing a tumor antigen or fragment, variant, or derivative thereof.
b) With at least one histone stemloop,
c) Resolved by the nucleic acid sequence of the invention comprising or encoding a poly (A) sequence or a polyadenylation signal.

Alternatively, any suitable stem-loop sequence other than the histone stem-loop sequence (derived from histone genes, especially the histone genes of the H1, H2A, H2B, H3, and H4 families), in all aspects and embodiments of the invention. You may use it.

The following figures are intended to further illustrate the invention and should not be construed as limiting the invention to these.
FIG. 1 shows the consensus sequences of histone stem-loops found from the stem-loop sequences of metazoans and protozoa (Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14 (1), 1-10. Doi: 10.261 / rna.782308). 4,001 histone stem-loop sequences from metazoans and protozoa were aligned and the number of nucleotides present was shown for all positions of the stem-loop sequence. The found consensus sequences representing the nucleotides present in all the analyzed sequences are described using the one-letter notation of nucleotides. In addition to the consensus sequence, sequences representing nucleotides present in at least 99%, 95%, and 90% of the analyzed sequences are shown. FIG. 2 shows the consensus sequence of histone stem-loops found from protozoan stem-loop sequences (Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14). (1), 1-10. Doi: 10.261 / rna.782308). 131 histone stem-loop sequences from protozoa were aligned and the number of nucleotides present was shown for all positions in the stem-loop sequence. The found consensus sequences representing the nucleotides present in all the analyzed sequences are described using the one-letter notation of nucleotides. In addition to the consensus sequence, sequences representing nucleotides present in at least 99%, 95%, and 90% of the analyzed sequences are shown. FIG. 3 shows the consensus sequence of histone stem-loops found in metazoan stem-loop sequences (Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14). (1), 1-10. Doi: 10.261 / rna.782308). 3,870 histone stem-loop sequences from metazoans were aligned and the number of nucleotides present was shown for all positions of the stem-loop sequence. The found consensus sequences representing the nucleotides present in all the analyzed sequences are described using the one-letter notation of nucleotides. In addition to the consensus sequence, sequences representing nucleotides present in at least 99%, 95%, and 90% of the analyzed sequences are shown. FIG. 4 shows the consensus sequence of histone stem-loops found in vertebrate stem-loop sequences (Davila Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14). (1), 1-10. Doi: 10.261 / rna.782308). 1,333 histone stem-loop sequences from vertebrates were aligned and the number of nucleotides present was shown for all positions of the stem-loop sequence. The found consensus sequences representing the nucleotides present in all the analyzed sequences are described using the one-letter notation of nucleotides. In addition to the consensus sequence, sequences representing nucleotides present in at least 99%, 95%, and 90% of the analyzed sequences are shown. FIG. 5 shows the consensus sequence of histone stem-loops found in human stem-loop sequences (Davilla Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14 ( 1), 1-10. Doi: 10.261 / rna.782308). 84 histone stem-loop sequences of human origin were aligned and the number of nucleotides present was shown for all positions of the stem-loop sequence. The found consensus sequences representing the nucleotides present in all the analyzed sequences are described using the one-letter notation of nucleotides. In addition to the consensus sequence, sequences representing nucleotides present in at least 99%, 95%, and 90% of the analyzed sequences are shown. Figures 6-21 show mRNA obtained from in vitro transcription. The names and sequences of mRNA obtained by in vitro transcription are described. The following abbreviations are used. ppLuc (GC): GC-rich mRNA sequence encoding the Kita-American firefly (Photinus pyraris) luciferase ag: 3'untranslated region (UTR) of the alphaglobin gene A64: Poly (A) sequence containing 64 adenilates A120: Poly (A) sequence containing 120 adenilates Histon SL: Histon stemloop aCPSL: Stemloop selected from the library for specific binding of αCP-2KL protein Polyo CL: 5'clover derived from poliovirus genomic RNA Leaf G30: Poly (G) sequence containing 30 guanylates U30: 30 Poly (U) sequence containing urizilate SL: Non-specific / artificial stem-loop N32: Non-specific sequence of 32 nucleotides NY-ESO -1 (G / C): GC-rich mRNA sequence encoding the human tumor antigen NY-ESO-1 Survival (G / C): GC-rich mRNA sequence encoding the human tumor antigen survivin MAGE-C1 (G) / C): In the GC-rich mRNA sequence encoding the human tumor antigen MAGE-C1, the following elements are emphasized: coding region (ORF) (capital), ag (bold), histon SL (underline), test Yet another sequence (Italic form). FIG. 6 shows the mRNA sequence of ppLuc (GC) -ag (SEQ ID NO: 43). By linearizing the original vector at the restriction enzyme site immediately after alphaglobin 3'-UTR (ag), mRNA having no poly (A) sequence is obtained. FIG. 7 shows the mRNA sequence of ppLuc (GC) -ag-A64 (SEQ ID NO: 44). By linearizing the original vector at the restriction enzyme site immediately after the A64 poly (A) sequence, mRNA terminating with the A64 poly (A) sequence is obtained. FIG. 8 shows the mRNA sequence of ppLuc (GC) -ag-histone SL (SEQ ID NO: 45). The A64 poly (A) sequence was replaced with histone SL. The histone stem-loop sequence used in the examples was described in Cakmacci et al. (2008). Obtained from Molecular and Cellular Biology , 28 (3), 1182-1194. FIG. 9 shows the mRNA sequence of ppLuc (GC) -ag-A64-histone SL (SEQ ID NO: 46). Histone SL was added to 3'of A64 poly (A). FIG. 10 shows the mRNA sequence of ppLuc (GC) -ag-A120 (SEQ ID NO: 47). The A64 poly (A) sequence was replaced with the A120 poly (A) sequence. FIG. 11 shows the mRNA sequence of ppLuc (GC) -ag-A64-ag (SEQ ID NO: 48). A second alpha globin 3'-UTR was added to the 3'of the A64 poly (A). FIG. 12 shows the mRNA sequence of ppLuc (GC) -ag-A64-aCPSL (SEQ ID NO: 49). A stem loop was added to the 3'of the A64 poly (A). Stem-loops were selected from the library for specific binding of the αCP-2KL protein (Thisted et al., (2001), The Journal of Biological Chemistry, 276 (20), 17484-17496). αCP-2KL is an isoform of αCP-2 and is the most strongly expressed αCP protein (alphaglobin mRNA poly (C) binding protein) (Makeyev et al., (2000), Genomics, 67 (3), 301-316), a group of RNA-binding proteins that bind to alphaglobin 3'-UTR (Chkheidze et al., (1999), Molecular and Cellular Biology, 19 (7), 4572-4581). FIG. 13 shows the mRNA sequence of ppLuc (GC) -ag-A64-polio CL (SEQ ID NO: 50). A 5'clover leaf derived from poliovirus genomic RNA was added to 3'of A64 poly (A). FIG. 14 shows the mRNA sequence of ppLuc (GC) -ag-A64-G30 (SEQ ID NO: 51). Thirty guanylate elongations were added to 3'of A64 poly (A). FIG. 15 shows the mRNA sequence of ppLuc (GC) -ag-A64-U30 (SEQ ID NO: 52). Thirty urizilate elongations were added to 3'of A64 poly (A). FIG. 16 shows the mRNA sequence of ppLuc (GC) -ag-A64-SL (SEQ ID NO: 53). A stem loop was added to the 3'of the A64 poly (A). The upper part of the stem and loop was removed (Babendure et al., (2006), RNA (New York, NY), 12 (5), 851-861). The stem loop consists of a 17 base pair length CG rich stem and a 6 base pair length loop. FIG. 17 shows ppLuc (GC) -ag-A64-N32 (SEQ ID NO: 54). Linearization of the original vector at another restriction enzyme site gives the mRNA with the poly (A) sequence followed by 32 additional nucleotides. FIG. 18 shows the mRNA sequence of NY-ESO-1 (GC) -ag-A64-C30 (SEQ ID NO: 55). FIG. 19 shows the mRNA sequence of NY-ESO-1 (GC) -ag-A64-C30-histone SL (SEQ ID NO: 56). FIG. 20 shows the mRNA sequence of survivin (GC) -ag-A64-C30-histone SL (SEQ ID NO: 57). FIG. 21 shows the mRNA sequence of MAGE-C1 (GC) -ag-A64-C30-histone SL (SEQ ID NO: 58). FIG. 22 shows that the combination of poly (A) and histone SL synergistically increases protein expression from mRNA. The effects of the poly (A) sequence, histone SL, and the combination of poly (A) and histone SL on luciferase expression from mRNA were investigated. Therefore, various mRNAs were electroporated into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after transfection. A small luciferase that has neither a poly (A) sequence nor histone SL is expressed from the mRNA. Both the poly (A) sequence or histone SL increase luciferase levels. However, surprisingly, the combination of poly (A) and histone SL acts synergistically as it increased luciferase levels many times more strongly than those found in any of the individual elements. For triplicate transfections, graph the data as mean RLU ± SD (relative optical units ± standard deviation). Specific RLUs are summarized in Example 14.2. FIG. 23 shows that the combination of poly (A) and histone SL increases protein expression from mRNA regardless of its order. The effects of the poly (A) sequence, histone SL, the combination of poly (A) and histone SL on luciferase expression from mRNA, and their order were investigated. Therefore, various mRNAs were lipofected into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after the start of transfection. Both the A64 poly (A) sequence or histone SL produce comparable luciferase levels. Increasing the length of the poly (A) sequence from A64 to A120 or A300 results in a moderate increase in luciferase levels. In contrast, the combination of poly (A) and histone SL increases luciferase levels much more than the extension of the poly (A) sequence. The combination of poly (A) and histone SL acts synergistically as it increased luciferase levels many times more strongly than those found in any of the individual elements. In A64-histone SL or histone SL-A250 mRNA, a synergistic effect of the combination of poly (A) and histone SL is observed regardless of the order of poly (A) and histone SL and the length of poly (A). For triplicate transfections, graph the data as mean RLU ± SD. Specific RLUs are summarized in Example 14.3. FIG. 24 shows that the increase in protein expression by the combination of poly (A) and histone SL is specific. The effect of combining poly (A) with histone SL or poly (A) with another sequence on luciferase expression from mRNA was investigated. Therefore, various mRNAs were electroporated into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after transfection. Both the poly (A) sequence or histone SL produce comparable luciferase levels. The combination of poly (A) and histone SL acts synergistically as it increased luciferase levels many times more strongly than those found in any of the individual elements. In contrast, the combination of poly (A) with any of the other sequences has no effect on luciferase compared to mRNA containing only the poly (A) sequence. Therefore, the combination of poly (A) and histone SL acts specifically and synergistically. For triplicate transfections, graph the data as mean RLU ± SD. Specific RLUs are summarized in Example 14.4. FIG. 25 shows that the combination of poly (A) and histone SL synergistically increases protein expression from mRNA in vivo. The effects of the poly (A) sequence, histone SL, and the combination of poly (A) and histone SL on luciferase expression from mRNA in vivo were investigated. Therefore, various mRNAs were injected intradermally into mice. Mice were sacrificed 16 hours after injection and luciferase levels at the injection site were measured. Luciferase is expressed from mRNA having either histone SL or poly (A) sequence. However, surprisingly, the combination of poly (A) and histone SL acts synergistically as it increased luciferase levels many times more strongly than those found in any of the individual elements. Graph the data as average RLU ± SEM (relative light unit ± average of standard error). Specific RLUs are summarized in Example 14.5. FIG. 26 shows that the combination of poly (A) and histone SL increases NY-ESO-1 protein expression from mRNA. The effect of the poly (A) sequence on NY-ESO-1 expression from mRNA and the combination of poly (A) and histone SL was investigated. Therefore, various mRNAs were electroporated into HeLa cells. NY-ESO-1 levels were measured 24 hours after transfection by flow cytometry. NY-ESO-1 is expressed from mRNA that has only the poly (A) sequence. However, surprisingly, the combination of poly (A) and histone SL strongly increases NY-ESO-1 levels many times higher than those found in the poly (A) sequence alone. Graph the data as a number relative to the fluorescence intensity. The central fluorescence intensity (MFI) is summarized in Example 14.6. FIG. 27 shows that the combination of poly (A) and histone SL increases the level of antibody induced by mRNA vaccination. The effect of the poly (A) sequence on the induction of anti-NY-ESO-1 antibody induced by mRNA vaccination and the combination of poly (A) and histone SL was investigated. Therefore, C57BL / 6 mice were vaccinated intradermally with various mRNAs complexed with protamine. Levels of NY-ESO-1 specific antibody in vaccinated and control mice were analyzed by ELISA with serial dilutions of serum. Anti-NY-ESO-1 IgG2a [b] is induced by mRNA having only the poly (A) sequence. However, surprisingly, the combination of poly (A) and histone SL increases anti-NY-ESO-1 IgG2a [b] levels many times higher than those found in the poly (A) sequence alone. Graph the data as the average endpoint titer. The average endpoint titer is summarized in Example 14.7.

In this regard, the nucleic acids of the invention according to the first aspect of the invention are preferably at least partially made or isolated nucleic acids by synthesis of DNA or RNA as described herein. It is particularly preferable to have.

The present invention is found in nature that the combination of a poly (A) sequence or polyadenylation signal with at least one histone stem-loop increases protein expression many times over the levels found in any of the individual elements. It is based on our surprising finding that it acts synergistically, despite representing another mechanism. The synergistic effect of the combination of poly (A) and at least one histone stem-loop is seen regardless of the order of poly (A) and histone stem-loops and the length of the poly (A) sequence.

Thus, the nucleic acid sequences of the invention are a) a coding region encoding at least one peptide or protein comprising a tumor antigen or fragment, variant or derivative thereof, and (preferably the encoded peptide or protein). It is particularly preferred that it comprises or encodes) b) at least one histone stemloop and c) a poly (A) sequence or a polyadenylated sequence to increase expression levels. In some embodiments, the encoded protein retains a histone protein, particularly a histone protein of the H4, H3, H2A, and / or H2B histone family, or histone (like) function, i.e. a function of forming a nucleosome. It may be preferable that it is not a fragment, derivative, or variant of the protein. Also, the encoded protein typically does not correspond to a histone linker protein of the H1 histone family. Nucleic acid molecules of the invention typically do not contain any regulatory signal (in the mouse histone gene, in particular the mouse histone gene H2A, more preferably in the mouse histone gene H2A614 5'and / or in particular 3'). In particular, the nucleic acid molecule of the present invention does not contain a mouse histone gene, in particular a histone stem loop and / or a histone stem loop processing signal derived from the mouse histone gene H2A, most preferably the mouse histone gene H2A614.

Also, the nucleic acids of the invention typically, in element (a), reporter proteins (eg, luciferase, GFP, EGFP, β-galactosidase, especially EGFP), galactokinase (galK) and / or markers or selective proteins. (For example, alphaglobin, galactokinase, and xanthin: guanine phosphoribosyl transferase (GPT)), or bacterial reporter protein (eg, chloramphenicol acetyl transferase (CAT)), or other bacterial antibiotic resistant proteins (eg For example, it does not provide an antibiotic resistant protein derived from the bacterial neo gene).

It is understood that the reporter, marker, or selected protein is typically not the tumor antigen according to the invention. Reporters, markers, or selective proteins, or their underlying genes, are commonly used as research tools in bacteria, cell cultures, animals, or plants. These impart measurable or easily identifiable properties to the organism expressing it (preferably heterologously), allowing measurement or selection. Specifically, the marker or selected protein exhibits a selectable function. Typically, such selectable, marker, or reporter proteins are not naturally present in humans or other mammals and are derived from other organisms, especially bacteria or plants. Therefore, a protein having a selection, marker, or reporter function derived from a species other than mammals, particularly a species other than humans, is preferably understood to be a protein having the property of acting as a "tumor antigen" according to the present invention. Is excluded from. Specifically, selectable, marker or reporter proteins allow transformed cells to be identified, for example, by fluorescence or other spectroscopic techniques, and in vitro assays based on resistance to antibiotics. Therefore, a selectable, reporter, or marker gene that imparts such properties to transformed cells is not understood to be a protein that typically acts as a tumor antigen according to the invention in vivo.

In either case, the reporter, marker, or selective protein usually exerts no tumor antigenicity and therefore no immunological effect in treating the tumor disease. Even if any single reporter, marker, or selected protein exerts an antigenic effect (in addition to its reporter, marker, or marker function), such reporter, marker, or selected protein is preferably preferred. It is not understood to be a "tumor antigen" within the meaning of the present invention.

In contrast, proteins or peptides that act as tumor antigens according to the invention, except in particular the H1, H2A, H2B, H3, and H4 family histone genes, typically have a selection, marker, or reporter function. Not shown. Even if any single "tumor antigen" exhibits selectable, marker, or reporter function (in addition to its tumor antigen function), such tumor antigen is preferably within the meaning of the present invention. Not understood to be a "selection, marker, or reporter protein".

Most preferably, the proteins that act as tumor antigens according to the invention are derived from mammals, especially humans, especially mammalian tumors, and are understood to be unsuitable as selectable, marker, or reporter proteins. Specifically, such tumor antigens are derived from mammalian, especially human tumors. These tumor antigenic proteins are understood to be antigenic because the tumor antigenic protein causes the subject's immune system to fight the subject's tumor cells by a TH1 and / or TH2 immune response. To be able to treat the subject by inducing an immune response of the subject. Thus, such antigenic tumor proteins are typically mammalian, especially human, proteins that characterize the subject's cancer type.

Therefore, it is preferred that (a) the coding region encoding at least one peptide or protein is heterologous to at least (b) at least one histone stemloop, or more broadly, any suitable stemloop. .. In other words, "heterologous" in the present invention means that at least one stem-loop sequence encodes a (tumor antigen) protein or peptide of the nucleic acid element (a) of the present invention (eg, 3). It means that it does not exist in nature as a (regulatory) sequence (in the UTR). Therefore, the (histone) stem-loop of the nucleic acid of the present invention is preferably derived from the 3'UTR of a gene other than the gene containing the coding region of the nucleic acid element (a) of the present invention. For example, the coding region of element (a) does not encode a histone protein, or a fragment, variant, or derivative thereof (which retains the function of the histon protein) when the nucleic acids of the invention are heterologous. Encode any other peptide or sequence (of the same or different species) that exerts a tumor antigenic function other than biological function, preferably histone (like) function, eg, (eg, mammals, especially humans). Encodes a protein (which exerts a tumor antigenic function in terms of vaccination against a tumor) to elicit an immune response against a subject's tumor cells (preferably expressing the tumor antigen encoded by the nucleic acid of the invention). ..

In this regard, the nucleic acids of the invention are in the 5'to 3'direction,
a) A coding region encoding at least one peptide or protein containing a tumor antigen or a fragment, variant, or derivative thereof.
b) With at least one histone stemloop optionally having no histone downstream element (HDE) at 3'of the histone stemloop.
c) It is particularly preferred to include or encode a poly (A) sequence or a polyadenylation signal.

The term "histone downstream element (HDE)" represents the binding site of U7snRNA involved in the processing of histone pre-mRNA to mature histone mRNA, from about 15 to about 20 nucleotides in the naturally occurring histone stem-loop 3'. Refers to the purin-rich polynucleotide elongation of. For sea urchins, for example, the HDE is CAAGAAAGA (Dominski, Z. and WF Marzluff (2007), Gene 396 (2): 373-90).

Furthermore, the nucleic acid of the present invention according to the first aspect of the present invention preferably does not contain an intron.

In another particularly preferred embodiment, the nucleic acid sequence of the invention according to the first aspect of the invention is from 5'to 3'a) preferably a tumor antigen or fragment, variant or derivative thereof. With a coding region encoding at least one peptide or protein containing
c) Poly (A) sequence and
b) Includes or encodes with at least one histone stemloop.

The nucleic acid sequence of the present invention according to the first embodiment of the present invention is, for example, any (single-stranded or double-stranded) DNA (preferably genomic DNA, plasmid DNA, single-stranded DNA molecule, double-stranded). It may be selected from (but not limited to, DNA molecules, etc.), for example, from any PNA (peptide nucleic acid), or, for example, any (single or double) RNA (preferably). Includes any suitable nucleic acid that may be selected from messenger RNA (mRNA). In addition, the nucleic acid sequence of the present invention may contain viral RNA (vRNA). However, the nucleic acid sequence of the present invention does not have to be viral RNA and may not contain viral RNA. More specifically, the nucleic acid sequences of the invention do not contain viral sequence elements such as viral enhancers or viral promoters (eg, inactivated viral promoters or sequence elements, and more specifically are not inactivated by substitution strategies. ), Or it may not contain other viral sequence elements or viral or retroviral nucleic acid sequences. More specifically, the nucleic acid sequence of the present invention does not have to be a retrovirus or viral vector, or a modified retrovirus or viral vector.

In any case, the nucleic acid sequence of the present invention may or may not contain an enhancer and / or promoter sequence, and the enhancer and / or promoter sequence may be modified. It may not be modified, it may be activated, or it may not be activated. The enhancer or promoter sequence may or may not be expressible in plants and / or may or may not be expressible in eukaryotes and / or prokaryotes. It may or may not be expressible in an organism. The nucleic acid sequence of the present invention may or may not contain a sequence encoding a (self-splicing) ribozyme.

In a specific embodiment, the nucleic acid sequence of the present invention may or may not contain self-splicing RNA (replicon).

Preferably, the nucleic acid sequence of the present invention is plasmid DNA, or RNA, especially mRNA.

In a particular embodiment of the first aspect of the invention, the nucleic acid of the invention is a nucleic acid contained in a nucleic acid suitable for in vitro transcription, particularly in a suitable in vitro transcription vector (eg, T3, T7, or Sp6 promoter). A plasmid or linear nucleic acid sequence containing a specific promoter for in vitro transcription, etc.).

In a further particularly preferred embodiment of the first aspect of the invention, the nucleic acid of the invention is an expression system (eg, in an expression vector or plasmid), particularly a prokaryote (eg, a bacterium such as Escherichia coli) or a eukaryote (eg, a bacterium such as E. coli). For example, it is contained in a nucleic acid suitable for transcription and / or translation in an expression system of mammalian cells such as CHO cells, yeast cells, or insect cells, or whole organisms such as plants or animals.

The term "expression system" means a system (cell culture or whole organism) suitable for the production (recombinant expression) of peptides, proteins, or RNAs, especially mRNAs.

The nucleic acid sequence of the invention according to the first aspect of the invention comprises or encodes at least one histone stemloop. In the present invention, such histone stem-loops (whether or not they are histone stem-loops) are generally typically derived from the histone gene and are two adjacent, wholly or partially inversely complementary. Sequences include those in which stem loops are formed by intramolecular base pairing. Stem-loops can be formed in single-stranded DNA, or more generally RNA. The structure is also known as a hairpin or hairpin loop, usually consisting of a stem and a (terminal) loop in a continuous array, the stem being a short array as a type of spacer that constitutes a loop of stem-loop structure. It is formed by two adjacent, wholly or partially inversely complementary sequences, which are partitioned by. The two adjacent, wholly or partially inversely complementary sequences can be defined, for example, as stem-loop elements, stem 1 and stem 2. Stem-loops are formed in a continuous sequence when these two adjacent, wholly or partially inversely complementary sequences (eg, stem-loop elements, stem 1 and stem 2) base pair with each other. It results in double-stranded nucleic acid sequence extension with an unpaired loop at the end formed by a stem-loop element, a short sequence located between stem 1 and stem 2. Thus, an unpaired loop typically represents a region of nucleic acid that cannot base pair with any of these stem-loop elements. The resulting lollipop structure is an important component of many RNA secondary structures. Therefore, the formation of a stem-loop structure depends on the stability of the resulting stem and loop region, and the first requirement is typically a sequence of sequences that can be folded to form a paired double helix. Existence. The stability of paired stem-loop elements depends on the length of the pairing region, the number of mismatches or bulges contained (especially for long double-stranded extensions, a small number of mismatches are typically acceptable), and Determined by base composition. In the present invention, a loop length of 3 to 15 bases can be considered, but a more preferable loop length is 3 to 10 bases, more preferably 3 to 8 bases, 3 bases to 7 bases, 3 bases to 6 bases, Or even more preferably 4 to 5 bases, most preferably 4 bases. The stem sequence forming the double-stranded structure typically has a length of 5 to 10 bases, more preferably 5 to 8 bases.

In the present invention, histone stemloops are typically derived from histone genes (eg, genes from the histone families H1, H2A, H2B, H3, H4) and two adjacent, wholly or partially inverted. Complementary sequences include those formed by intramolecular base pairing to form a stem loop. Typically, the histone 3'UTR stemloop is an RNA element involved in the nuclear cytoplasmic transport of histone mRNAs, as well as the regulation of stability and transport efficiency in the cytoplasm. The mRNA of the histone gene in metazoans does not contain polyadenylation and poly (A) tail, but instead has a highly conserved stem-loop and a purin-rich region approximately 20 nucleotides downstream (downstream histone element, or HDE). 3'processing is performed at the site between and. The histone stemloop is bound by a 31 kDa stemloop binding protein (also called SLBP, histone hairpin binding protein or HBP). Such histone stem-loop structures are preferably used in the present invention in combination with other sequence elements and structures, wherein the other sequence elements and structures are in nature (which is an untransformed survivor / cell). Is not present in the histone gene, but is combined according to the present invention to provide an artificial heterologous nucleic acid. Accordingly, the present invention specifically comprises engineerable and / or regulatory sequence regions (transcription and regulatory) of genes that are not present in histone genes or histone genes in metazoans and that encode proteins other than histones. It is based on the finding that artificial (non-native) combinations of histone stem-loop structures with other heterologous sequence elements isolated from (/ or affect translation) have a beneficial effect. Therefore, one aspect of the invention provides a combination of a histone stem-loop structure and a poly (A) sequence or a sequence representing a polyadenylation signal (3'end of the coding region) that is not present in the histone gene of metazoans. do. According to another preferred embodiment of the invention, a combination of a histone stem-loop structure and a coding region encoding a tumor antigen protein preferably not present in the histone gene of metazoans is provided with this (coding region and histone). It is different from the stem-loop sequence). Such tumor antigen proteins are preferably present in mammals, preferably humans, when suffering from a tumor disease. In a more preferred embodiment, all elements (a), (b), and (c) of the nucleic acids of the invention are heterologous to each other and are artificially combined from three different sources (eg, (a) human). Gene-derived tumor antigen protein coding regions, (b) metazoans, such as mammalian, non-human or untranslated regions of human histone genes, and (c), for example, genes other than histone genes and Poly (A) sequence or polyadenylation signal derived from the untranslated region of a gene other than the untranslated region of the tumor antigen coding region according to the nucleic acid element (a) of the present invention).

Thus, histone stem-loops are the stem-loop structures described herein, which are their naturally binding partners, stem-loop binding proteins (SLBPs, histone hairpin bindings), where it is preferred to be functionally defined. It exhibits / retains the property of binding to proteins (also called HBPs).

According to the present invention, the histone stem-loop sequence according to the component (b) according to claim 1 does not have to be derived from the mouse histone protein. More specifically, the histone stem-loop sequence does not have to be derived from the mouse histone gene H2A614. In addition, the nucleic acid of the present invention does not have to contain the mouse histone stem-loop sequence or the mouse histone gene H2A614. Furthermore, even if the nucleic acid sequence of the invention may contain at least one mammalian histone gene, the nucleic acid sequence of the invention is a stem-loop processing signal, more specifically a mouse histone processing signal, most specifically. It does not have to contain the mouse stem-loop processing signal H2kA614. However, the at least one mammalian histone gene does not have to be SEQ ID NO: 7 as described in WO 01/12824.

式（ＩＩ）（ステム境界エレメントを含むステムループ配列）：

（式中、
ステム１又はステム２の境界エレメントＮ_１−６は、１個〜６個、好ましくは２個〜６個、より好ましくは２個〜５個、更により好ましくは３個〜５個、最も好ましくは４個〜５個、又は５個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、
ステム１［Ｎ_０−２ＧＮ_３−５］は、エレメントステム２に対して逆相補的であるか又は部分的に逆相補的であり、５個〜７個のヌクレオチドの連続配列であり、Ｎ_０−２は、０個〜２個、好ましくは０個〜１個、より好ましくは１個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、Ｎ_３−５は、３個〜５個、好ましくは４個〜５個、より好ましくは４個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、Ｇは、グアノシン又はそのアナログであり、任意でシチジン又はそのアナログによって置換されてもよいが、但し、その場合、ステム２におけるその相補的ヌクレオチドであるシチジンも、グアノシンによって置換され、
ループ配列［Ｎ_０−４（Ｕ／Ｔ）Ｎ_０−４］は、エレメントステム１とステム２との間に位置し、３個〜５個のヌクレオチド、より好ましくは４個のヌクレオチドの連続配列であり、各Ｎ_０−４は、互いに独立して、０個〜４個、好ましくは１個〜３個、より好ましくは１個〜２個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、Ｕ／Ｔは、ウリジン又は任意でチミジンを表し、
ステム２［Ｎ_３−５ＣＮ_０−２］は、エレメントステム１に対して逆相補的であるか又は部分的に逆相補的であり、５個〜７個のヌクレオチドの連続配列であり、Ｎ_３−５は、３個〜５個、好ましくは４個〜５個、より好ましくは４個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、Ｎ_０−２は、０個〜２個、好ましくは０個〜１個、より好ましくは１個のＮの連続配列であり、各Ｎは、互いに独立して、Ａ、Ｕ、Ｔ、Ｇ、及びＣから選択されるヌクレオチド、又はこれらのヌクレオチドアナログから選択され、Ｃは、シチジン又はそのアナログであり、任意でグアノシン又はそのアナログによって置換されてもよいが、但し、その場合、ステム１におけるその相補的ヌクレオチドであるグアノシンも、シチジンによって置換され、
ステム１及びステム２は、互いに塩基対合して逆相補的配列を形成することができ、前記塩基対合は、例えば、ヌクレオチドＡとＵ／Ｔ又はＧとＣとのワトソン−クリック塩基対合、又は非ワトソン−クリック塩基対合（例えば、ゆらぎ塩基対合、逆ワトソン−クリック塩基対合、フーグスティーン塩基対合、逆フーグスティーン塩基対合）によってステム１とステム２との間で生じ得るか、或いは、互いに塩基対合して部分的に逆相補的な配列を形成することができ、この場合、１つのステムにおける１以上の塩基が、他のステムの逆相補的な配列に相補的塩基を有しないことに基づいて、ステム１とステム２との間で不完全な塩基対合が生じ得る）。 According to one preferred embodiment of the first aspect of the invention, the nucleic acid sequence of the invention is at least one histone stemloop sequence, preferably at least one of the following formulas (I) or (II). Contains or encodes one of the histone stem-loop sequences:
Equation (I) (Stem-loop array without stem boundary elements):

Formula (II) (stem loop array including stem boundary elements):

(During the ceremony,
_{The boundary elements N 1-6} of the stem 1 or the stem 2 are 1 to 6, preferably 2 to 6, more preferably 2 to 5, still more preferably 3 to 5, and most preferably. A contiguous sequence of 4-5 or 5 N, each N independently selected from nucleotides selected from A, U, T, G, and C, or nucleotide analogs thereof. ,
Stem 1 [N _0-2 GN _3-5 ] is reverse-complementary or partially reverse-complementary to element stem 2 and is a contiguous sequence of 5-7 nucleotides, N. _0-2 is a continuous sequence of 0 to 2, preferably 0 to 1, more preferably 1 N, and each N is independent of each other, A, U, T, G, and. Nucleotides selected from C, or selected from these nucleotide analogs, N _3-5 is a contiguous sequence of 3-5, preferably 4-5, more preferably 4 N, each. N is independently selected from nucleotides selected from A, U, T, G, and C, or nucleotide analogs thereof, where G is guanosine or an analog thereof, optionally by cytidine or an analog thereof. It may be substituted, provided that cytidine, its complementary nucleotide in stem 2, is also substituted with guanosine.
The loop sequence [N _0-4 (U / T) N _0-4 ] is located between the element stem 1 and stem 2, and is a continuous sequence of 3 to 5 nucleotides, more preferably 4 nucleotides. Each N _0-4 is a continuous sequence of 0-4, preferably 1-3, more preferably 1-2 N, independent of each other, with each N being mutually exclusive. Independently selected from nucleotides selected from A, U, T, G, and C, or nucleotide analogs thereof, U / T represents uridine or optionally thymidine.
Stem 2 [N _3-5 CN _0-2 ] is reverse-complementary or partially reverse-complementary to element stem 1 and is a contiguous sequence of 5-7 nucleotides, N. _3-5 is a continuous sequence of 3-5, preferably 4-5, more preferably 4 N, each N being independent of each other, A, U, T, G, and Nucleotides selected from C, or selected from these nucleotide analogs, N _0-2 is a contiguous sequence of 0 to 2, preferably 0 to 1, more preferably 1 N, each. N is independently selected from nucleotides selected from A, U, T, G, and C, or nucleotide analogs thereof, and C is cytidine or an analog thereof, optionally by guanosine or an analog thereof. It may be substituted, provided that guanosine, its complementary nucleotide in stem 1, is also substituted with cytidine.
Stem 1 and stem 2 can be base paired with each other to form an inverse complementary sequence, wherein the base pairing is, for example, Watson-Crick base pairing of nucleotides A and U / T or G and C. , Or by non-Watson-Crick base pairing (eg, fluctuating base pairing, reverse Watson-Crick base pairing, Hoogsteen base pairing, reverse Hugsteen base pairing) between stem 1 and stem 2. It can occur or can be base paired with each other to form partially inverse complementary sequences, in which case one or more bases in one stem become inverse complementary sequences in another stem. Incomplete base pairing can occur between stem 1 and stem 2 due to the lack of complementary bases).

In connection with the above, wobble base pairing is typically a non-Watson-click base pairing between two nucleotides. The four major wobble base pairs (which can be used) in this context are guanosine-uridine, iosin-uridine, iosin-adenosine, iosin-cytidine (GU / T, I-U / T, IA,). And IC), and adenosine-cytidine (AC).

Therefore, in the present invention, a wobble base is a base that forms a wobble base pair with another base as described above. Therefore, non-Watson-click base pairing, such as wobble base pairing, can occur in the stem of the histone stem-loop structure according to the present invention.

In connection with the above, the partially inversely complementary sequence contains up to two, preferably only one, mismatch in the stem structure of the stem loop sequence formed by base pairing of stem 1 and stem 2. In other words, Stem 1 and Stem 2 are preferably (100% accurate) base paired (100% possible exact Watson-click base pairing or non-Watson-click) with each other over the entire sequence of Stem 1 and Stem 2. Base pairing) can be formed to form inverse complementary sequences, where each base has an exact Watson-Crick base pendant or non-Watson-Crick base pendant as a complementary binding partner. Alternatively, Stem 1 and Stem 2 can preferably be partially base paired with each other over the entire sequence of Stem 1 and Stem 2, in this case 100% accurate Watson-Crick base pairing. Or at least about 70%, about 75%, about 80%, about 85%, about 90%, or about 95% of the non-Watson-Crick base pairing is the exact Watson-Crick base pairing or non-Watson. -The remaining bases, which are occupied by click base pairs and up to about 30%, about 25%, about 20%, about 15%, about 10%, or about 5%, are unpaired.

According to a preferred embodiment of the first aspect of the invention, at least one histon stem loop sequence (including the stem boundary element) of the nucleic acid sequence of the invention as defined herein is about 15 nucleotides in length to about 45. Nucleotide length, preferably about 15 nucleotides to about 40 nucleotides, preferably about 15 nucleotides to about 35 nucleotides, preferably about 15 nucleotides to about 30 nucleotides, and even more preferably about 20 nucleotides to about 30. It has a nucleotide length, most preferably about 24 nucleotides to about 28 nucleotides in length.

According to a more preferred embodiment of the first aspect of the invention, at least one histon stem loop sequence (not including the stem boundary element) of the nucleic acid sequence of the invention as defined herein is about 10 nucleotides in length. About 30 nucleotides in length, preferably about 10 nucleotides in length to about 20 nucleotides in length, preferably about 12 nucleotides in length to about 20 nucleotides in length, preferably about 14 nucleotides in length to about 20 nucleotides in length, and even more preferably about 16 nucleotides in length. It has a length of about 17 nucleotides, most preferably about 16 nucleotides.

式（ＩＩａ）（ステム境界エレメントを含むステムループ配列）：

（式中、Ｎ、Ｃ、Ｇ、Ｔ、及びＵは、上に定義した通りである）。 According to a more preferred embodiment of the first aspect of the present invention, the nucleic acid sequence of the present invention according to the first aspect of the present invention is at least one of the following specific formulas (Ia) or (IIa). May include or encode at least one histone stemloop sequence according to:
Equation (Ia) (Stemloop array without stem boundary elements):

Equation (IIa) (stem loop array including stem boundary elements):

(In the formula, N, C, G, T, and U are as defined above).

式（ＩＩｂ）（ステム境界エレメントを含むステムループ配列）：

（式中、Ｎ、Ｃ、Ｇ、Ｔ、及びＵは、上に定義した通りである）。 According to an even more preferred embodiment of the first aspect, the nucleic acid sequence of the present invention comprises at least one histone stemloop sequence according to at least one of the following specific formulas (Ib) or (IIb): Or may be coded:
Equation (IIb) (Stem-loop sequence without stem boundary elements):

Equation (IIb) (stem loop array including stem boundary elements):

(In the formula, N, C, G, T, and U are as defined above).

式（ＩＩｃ）（ステム境界エレメントを含む後生動物及び原生動物のヒストンステムループコンセンサス配列）：

式（Ｉｄ）（ステム境界エレメントを含まない）：

式（ＩＩｄ）（ステム境界エレメントを含む）：

式（Ｉｅ）（ステム境界エレメントを含まない原生動物のヒストンステムループコンセンサス配列）：

式（ＩＩｅ）（ステム境界エレメントを含む原生動物のヒストンステムループコンセンサス配列）：

式（Ｉｆ）（ステム境界エレメントを含まない後生動物のヒストンステムループコンセンサス配列）：

式（ＩＩｆ）（ステム境界エレメントを含む後生動物のヒストンステムループコンセンサス配列）：

式（Ｉｇ）（ステム境界エレメントを含まない脊椎動物のヒストンステムループコンセンサス配列）：

式（ＩＩｇ）（ステム境界エレメントを含む脊椎動物のヒストンステムループコンセンサス配列）：

式（Ｉｈ）（ステム境界エレメントを含まないヒト（Ｈｏｍｏ ｓａｐｉｅｎｓ）のヒストンステムループコンセンサス配列）：

式（ＩＩｈ）（ステム境界エレメントを含むヒトのヒストンステムループコンセンサス配列）：

上記式（Ｉｃ）〜（Ｉｈ）又は（ＩＩｃ）〜（ＩＩｈ）のそれぞれにおいて、Ｎ、Ｃ、Ｇ、Ａ、Ｔ、及びＵは、上に定義した通りであり、各Ｕは、Ｔで置換されてもよく、ステムエレメント１及び２におけるそれぞれの（高度に）保存されているＧ又はＣは、その相補的ヌクレオチド塩基であるＣ又はＧで置換されてもよいが、但し、その場合、対応するステムにおける相補的ヌクレオチドが、並行してその相補的ヌクレオチドによって置換され、及び／又はＧ、Ａ、Ｔ、Ｕ、Ｃ、Ｒ、Ｙ、Ｍ、Ｋ、Ｓ、Ｗ、Ｈ、Ｂ、Ｖ、Ｄ、及びＮは、以下の表に定義するヌクレオチド塩基である：

According to an even more preferred embodiment of the first aspect of the present invention, the nucleic acid sequence of the present invention according to the first aspect of the present invention represents a histone stem-loop sequence prepared in a stem-loop structure and according to Example 1. It may contain or encode at least one histone stemloop sequence according to at least one of the following specific formulas (Ic)-(Ih) or (IIc)-(IIh) represented as a linear sequence. :
Formula (Ic) (Histone stem-loop consensus sequence of metazoans and protozoa without stem boundary elements):

Formula (IIc) (Histone stem-loop consensus sequence of metazoans and protozoa including stem boundary elements):

Equation (Id) (not including stem boundary elements):

Equation (IId) (including stem boundary element):

Formula (Ie) (protozoan histone stem-loop consensus sequence without stem boundary elements):

Formula (IIe) (protozoan histone stem-loop consensus sequence containing stem boundary elements):

Formula (If) (histone stem-loop consensus sequence of metazoans without stem boundary elements):

Formula (IIf) (histone stem-loop consensus sequence of metazoans including stem boundary elements):

Equation (Ig) (Vertebrate histone stem-loop consensus sequence without stem boundary element):

Formula (IIg) (Vertebrate histone stem-loop consensus sequence containing stem boundary elements):

Equation (Ih) (Homo sapiens histone stem-loop consensus sequence without stem boundary elements):

Formula (IIh) (human histone stem-loop consensus sequence including stem boundary elements):

In each of the above formulas (Ic) to (Ih) or (IIc) to (IIh), N, C, G, A, T, and U are as defined above, and each U is replaced with T. The respective (highly) conserved Gs or Cs in stem elements 1 and 2 may be substituted with their complementary nucleotide bases, C or G, provided that the correspondence is made. Complementary nucleotides in the stem are replaced in parallel by the complementary nucleotides and / or G, A, T, U, C, R, Y, M, K, S, W, H, B, V, D and N are the nucleotide bases defined in the table below:

In this regard, the histone stem-loop sequence according to at least one of the formulas (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) of the present invention is particularly preferably. From naturally occurring histone stem-loop sequences, more particularly preferably from protozoan or metazoan histone stem-loop sequences, even more preferably from vertebrate histone stem-loop sequences, most preferably from mammalian histone stem-loop sequences. , Especially selected from human histone stem-loop sequences.

According to a particularly preferred embodiment of the first aspect, the histone stem according to at least one of the formulas (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh) of the present invention. Loop sequences are found in nature in metazoans and protozoans (Fig. 1), protozoa (Fig. 2), metazoans (Fig. 3), vertebrates (Fig. 4), and humans (Fig. 5) shown in FIGS. 1-5. A histon stem-loop sequence containing the most frequently occurring nucleotide of the existing histon stem-loop sequences, or the most frequently or second most frequently occurring nucleotide at each nucleotide position. In this regard, at least 80%, preferably at least 85%, or most preferably at least 90% of all nucleotides correspond to the most frequently occurring nucleotides of the naturally occurring histone stem-loop sequences. Is particularly preferred.

In a more specific embodiment of the first aspect, the histone stem-loop sequence according to at least one of the specific formulas (I) or (Ia)-(Ih) of the present invention is made according to Example 1. The following histone stem-loop sequences (not including stem boundary elements) that represent the histone stem-loop sequences are selected from:
VGYYYYHHTHRVVRCB (SEQ ID NO: 13 according to formula (Ic))
SGYYYTYTMARRCS (SEQ ID NO: 14 according to formula (Ic))
SGYYCTTTTMAGRRCS (SEQ ID NO: 15 according to formula (Ic))

DGNNNBNNTHVNNNCH (SEQ ID NO: 16 according to formula (Ie))
RGNNNYHBTHRDNNCY (SEQ ID NO: 17 according to formula (Ie))
RGNDBYHYTHRDHNCY (SEQ ID NO: 18 according to formula (Ie))

VGYYYYTYHTHRVRRCB (SEQ ID NO: 19 according to formula (If))
SGYYCTTYTMAGRRCS (SEQ ID NO: 20 according to formula (If))
SGYYCTTTTMAGRRCS (SEQ ID NO: 21 according to formula (If))

GGYYCTTYTHAGRRCC (SEQ ID NO: 22 according to formula (Ig))
GGCYCTTYTMAGRGCC (SEQ ID NO: 23 according to formula (Ig))
GGCTCTTTTMAGRGCC (SEQ ID NO: 24 according to formula (Ig))

DGHYCTDYTHASRRCC (SEQ ID NO: 25 according to formula (Ih))
GGCYCTTTTHAGRGCC (SEQ ID NO: 26 according to formula (Ih))
GGCYCTTTTMAGRGCC (SEQ ID NO: 27 according to formula (Ih))

In addition, in this regard, the following histone stem-loop sequences (including stem boundary elements) made according to Example 1 according to one of the specific formulas (II) or (IIa)-(IIh) Especially preferred:
H ^* H ^* HHVVGYYYYHHTHRVVRCBVHH ^* N ^* N ^* (SEQ ID NO: 28 according to formula (IIc))
M ^* H ^* MHMSGYYYTYTMARRRSCSMCH ^* H ^* H ^* (SEQ ID NO: 29 according to formula (IIc))
M ^* M ^* MMMSGYYCTTTTMAGRRCSACH ^* M ^* H ^* (SEQ ID NO: 30 according to formula (IIc))

N ^* N ^* NNNDGNNNNBNNTHVNNNCHNHN ^* N ^* N ^* (SEQ ID NO: 31 according to formula (IIe))
N ^* N ^* HHNRGNNNNYHBTHRDNNCYDHH ^* N ^* N ^* (SEQ ID NO: 32 according to formula (IIe))
N ^* H ^* HHVRGNDBYHYTHRDHNCYRHH ^* H ^* H ^* (SEQ ID NO: 33 according to formula (IIe))

H ^* H ^* MHMVGYYYYTYHTHRVRRCBVMH ^* H ^* N ^* (SEQ ID NO: 34 according to formula (IIf))
M ^* M ^* MMMSGYYCTTYTMAGRRRCSMCH ^* H ^* H ^* (SEQ ID NO: 35 according to formula (IIf))
M ^* M ^* MMMSGYYCTTTTMAGRRCSACH ^* M ^* H ^* (SEQ ID NO: 36 according to formula (IIf))

H ^* H ^* MAMGGYYCTTYTHAGRRCCVHN ^* N ^* M ^* (SEQ ID NO: 37 according to formula (IIg))
H ^* H ^* AAMGGCYCTTYTMAGRGCCVCH ^* H ^* M ^* (SEQ ID NO: 38 according to formula (IIg))
M ^* M ^* AAMGGCTCTTTTMAGRGCCMCY ^* M ^* M ^* (SEQ ID NO: 39 according to formula (IIg))

N ^* H ^* AAHDGHYCTDYTHASRRCCVHB ^* N ^* H ^* (SEQ ID NO: 40 according to formula (IIh))
H ^* H ^* AAMGGCYCTTTTHAGRGCCVMY ^* N ^* M ^* (SEQ ID NO: 41 according to formula (IIh))
H ^* M ^* AAAGGCYCTTTTMAGRGRCCRMY ^* H ^* M ^* (SEQ ID NO: 42 according to formula (IIh))

According to a more preferred embodiment of the first aspect of the present invention, the nucleic acid sequence of the present invention has a specific formula (I) or (Ia) to (Ih) or (II) or (IIa) to (IIh). Conserved (though not 100%) nucleotides or naturally occurring histone stem-loop sequences in at least one of these histone stem-loop sequences are at least about 80%, preferably at least about 85%, more preferably. Includes or encodes at least one histone stemloop sequence that exhibits at least about 90%, or even more preferably at least about 95% sequence identity.

In a preferred embodiment, the histone stem-loop sequence does not contain the loop sequence 5'-UUUC-3'. More specifically, the histone stemloops do not contain stem 1 sequence 5'-GGCUCU-3'and / or stem 2 sequence 5'-AGAGCC-3', respectively. In another preferred embodiment, the stem-loop sequence does not contain loop sequence 5'-CCUGCCC-3'or loop sequence 5'-UGAAU-3'. More specifically, the stem loops do not contain stem 1 sequence 5'-CCUGAGC-3' or stem 1 sequence 5'-ACCUUCUCCA-3', respectively, and / or stem 2 sequence 5'-. It does not contain GCUCAGG-3'or 5'-UGGAGAAAGGU-3'. Also, unless the invention is expressly limited to histone stem-loop sequences, the stem-loop sequences are preferably not derived from the 3'-untranslated region of the mammalian insulin receptor. Further, preferably, the nucleic acid of the present invention does not have to contain a histone stem-loop processing signal, and specifically, it does not have to be derived from the mouse histone gene H2A614 gene (H2kA614).

The nucleic acid sequence of the present invention according to the first aspect of the present invention may optionally contain or encode a poly (A) sequence. When comprising or encoding the poly (A) sequence, such poly (A) sequence is a sequence of about 25 to about 400 adenosine nucleotides, preferably a sequence of about 30 adenosine nucleotides, more preferably. Is a sequence of about 50 to about 400 adenosine nucleotides, more preferably a sequence of about 50 to about 300 adenosine nucleotides, even more preferably a sequence of about 50 to about 250 adenosine nucleotides, most preferably. It contains a sequence of about 60 to about 250 adenosine nucleotides. In this regard, the term "about" refers to a deviation of ± 10% of the value to which it is attached. Therefore, the poly (A) sequence contains at least 25 or more than 25, more preferably at least 30, more preferably at least 50 adenosine nucleotides. Therefore, such poly (A) sequences typically do not contain less than 20 adenosine nucleotides. More specifically, it does not contain 10 and / or less than 10 adenosine nucleotides.

Preferably, the nucleic acids according to the invention do not contain all or all but one or two or at least one or one of the components of the group consisting of: ribozymes (preferably self-splicing ribozymes). Encoding sequence, viral nucleic acid sequence, histone stem-loop processing signal (particularly histone stem-loop processing sequence derived from mouse histone H2A614 gene), Neo gene, inactivating promoter sequence, and inactivating enhancer sequence. Even more preferably, the nucleic acids according to the invention do not contain a ribozyme (preferably a self-splicing ribozyme) and one of the following groups: Neo gene, inactivating promoter sequence, inactivating enhancer sequence, histone stem. Loop processing signals (particularly histone stem-loop processing sequences derived from the mouse histone H2A614 gene). Thus, in a preferred embodiment, the nucleic acid may contain neither a ribozyme (preferably a self-splicing ribozyme) nor a Neo gene, or the ribozyme (preferably a self-splicing ribozyme) may be an arbitrary resistance gene (eg, usually of choice). It does not have to contain). In another preferred embodiment, the nucleic acids of the invention need not contain a ribozyme (preferably a self-splicing ribozyme) or a histone stem-loop processing signal (particularly a histone stem-loop processing sequence derived from the mouse histone H2A614 gene).

Alternatively, according to a first aspect of the invention, the nucleic acids of the invention are optionally specific protein factors (eg, cleavage and polyadenylation specific factors (CPSF), cleavage stimulating factors (CstF), cleavage factors I. And II (CF I and CF II), including polyadenylation signals as defined herein, as signals to polyadenylate (transcribed) mRNA by poly (A) polymerase (PAP)). In this regard, a consensus spore denylation signal comprising an NN (U / T) ANA consensus sequence is preferred. In a particularly preferred embodiment, the polyadenylation signal is the following sequence: AA (U / T) AAA or A (U / T) (U / T) AAA (in the formula, uridine is usually present in RNA and thymidine is , Usually present in DNA). In some embodiments, the polyadenylation signal used in the nucleic acids of the invention does not correspond to a polyadenylation processing signal derived from the U3 snRNA, U5, human gene G-CSF, or the SV40 polyadenylation signal sequence. Specifically, the polyadenylation signal is a gene of an arbitrary antibiotic resistance gene (or any other reporter or marker) as a gene in the coding region according to the element (a) of the nucleic acid of the present invention in the nucleic acid of the present invention. Alternatively, it cannot be combined with a select gene), especially a resistance neo gene (neomycin phosphotransferase). In addition, any of the above polyadenylation signals is preferably not combined with a histone stem-loop or histone stem-loop processing signal derived from the mouse histone gene H2A614 in the nucleic acid of the present invention.

The nucleic acid sequence of the present invention according to the first aspect of the present invention may further encode a protein or peptide containing a tumor antigen or a fragment, variant, or derivative thereof.

Tumor antigens are preferably located on the surface of (tumor) cells that characterize mammalian, especially human tumors (eg, systemic tumor disease or solid tumor disease). In addition, the tumor antigen may be selected from proteins that are overexpressed in tumor cells as compared to normal cells. In addition, tumor antigens also include antigens that are not denatured by themselves (or were originally denatured by themselves) but are expressed in cells associated with the putative tumor. Antigens associated with the tumor supply tube or its (re) formation, in particular antigens associated with angiogenesis (eg, growth factors such as VEGF, bFGF, etc.) are also included herein. Tumor-related antigens further include antigens derived from the cells or tissues in which the tumor is typically present. In addition, some substances (usually proteins or peptides) are expressed in patients suffering from cancer disease (whether or not they are aware of it), and these are concentrated in the body fluids of said patients. Exists in. These substances are also called "tumor antigens", but they are not antigens in a more limited sense than immune response inducers. Tumor antigens can be further classified into tumor-specific antigens (TSA) and tumor-related antigens (TAA). Only tumor cells can present TSA, not normal "healthy" cells. TSA typically results from tumor-specific mutations. TAA is more common and is usually presented by both tumor cells and healthy cells. Cytotoxic T cells can recognize these antigens and destroy antigen-presenting cells. In addition, tumor antigens may be present on the surface of the tumor, for example in the form of mutant receptors. In this case, the tumor antigen can be recognized by the antibody.

Further, tumor-related antigens can be classified into melanin-forming cell-specific antigens, cancer-testis antigens, and tissue-specific antigens also called tumor-specific antigens. Cancer-testis antigens are typically understood to be peptides or proteins of germline-related genes that can be activated in a wide range of tumors. Human cancer-testis antigens can be further subdivided into X-chromosome-encoded antigens (so-called CT-X antigens) and X-chromosome-uncoded antigens (so-called non-X CT antigens). The cancer-testis antigen encoded on the X chromosome includes, for example, a family of melanoma antigen genes (so-called MAGE family). Genes in the MAGE family can be characterized by a common MAGE homologous domain (MHD). These antigens, namely melanin-forming cell-specific antigens, cancer-testis antigens, and tumor-specific antigens, can elicit autologous and humoral immune responses, respectively. Therefore, the tumor antigen encoded by the nucleic acid sequence of the present invention is preferably a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen, and is preferably a CT-X antigen or a non-X CT. It may be an antigen, a binding partner of CT-X antigen, a binding partner of non-X CT antigen, or a tumor-specific antigen, more preferably a CT-X antigen, a binding partner of non-X CT antigen, or a tumor-specific antigen. It may be.

Particularly preferred tumor antigens are selected from the group consisting of: 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha- Actinin-4 / m, alpha-methylacyl coenzyme A racemase, ART-4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-cathepsin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, CA19-9, CA72-4, CA125, carreticrin, CAMEL, CASP-8 / m, cathepsin B, cathepsin L, CD19, CD20, CD22, CD25, CDE30, CD33 , CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, cathepsin-like protein, collagen XXIII type, COX-2, CT -9 / BRD6, Cten, Cyclin B1, Cyclin D1, cyp-B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3 , ETV6-AML1, EZH2, FGF-5, FN, Frau-1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP , GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, Hepsin, Her2 / neu, HERV-K-MEL, HLA-A ^* 0201-R17I, HLA-A11 / m, HLA-A2 / m , HNE, Homeobox NKX3.1, HOM-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL -13Ra2, IL-2R, IL-5, immature laminin receptor, calicrain 2, cathepsin 4, Ki67, KIAA0205, KIAA0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT , MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE -B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE-B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1 , MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, Mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, Substrate Protein 22, MC1R, M-CSF, ME1 / m, Mesoterin, MG50 / PXDN, MMP11, MN / CA IX antigen, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m , MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyl kinase-V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N -Ras / m, NSE, NY-ESO-B, NY-ESO-1, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, p15, p190 minor bcr -Abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 kinase, Pin-1, Pml / PARalpha, POTE, PRAME, PRDX5 / M, Prostain, Proteinase 3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SART -2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX-4, STAMP-1, STEP-1, Survival, Survival-2B, SYT-SSX -1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF beta, TGF beta RII, TGM-4, TPI / m, TRAG-3, TRG, TRP-1, TRP- 2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1, and WT1. Such tumor antigens are preferably p53, CA125, EGFR, Her2 / neu, hTERT, PAP, MAGE-A1, MAGE-A3, mesothelin, MUC-1, GP100, MART-1, tyrosinase, PSA, PSCA, PSMA, You may choose from the group consisting of STEAP-1, VEGF, VEGFR1, VEGFR2, Ras, CEA, or WT1, more preferably PAP, MAGE-A3, WT1, and MUC-1. Such antigens may preferably be selected from the group consisting of: MAGE-A1 (eg, MAGE-A1, accession number M77481), MAGE-A2, MAGE-A3, MAGE-A6 (eg, MAGE-A6). , Accession number NM_005363), MAGE-C1, MAGE-C2, melan-A (eg, melan-A, accession number NM_005511), GP100 (eg GP100, accession number M77348), tyrosinase (eg, tyrosinase, ac). Session number NM_000372), Survival g (eg Survival, accession number AF077350), CEA (eg CEA, accession number NM_004363), Her-2 / neu (eg Her-2 / neu, accession number M11730), WT1 (eg, WT1, accession number NM_000378), PRAME (eg, PRAME, accession number NM_006115), EGFRI (epithelial cell growth factor receptor 1) (eg, EGFRI (epithelial cell growth factor receptor 1), accession Number AF288738), MUC1, Mutin 1 (eg Mutin 1, Accession No. NM_002456), SEC61G (eg SEC61G, Accession No. NM_014302), hTERT (eg hTERT, Accession No. NM_198253), 5T4 (eg 5T4, Accession number NM_006670), TRP-2 (eg, TRP-2, accession number NM_001922), STEAP1, PCA, PSA, PSMA and the like.

Further, the tumor antigen may include an idiotype antigen associated with cancer or tumor disease, particularly lymphoma or lymphoma-related disease, said idiotype antigen being an immunoglobulin idiotype of lymphocytes or a T cell receptor idio of lymphocytes. The type.

Tumor antigen proteins for treating cancer or tumor disease are typically proteins of mammalian, preferably human, origin. The choice of the tumor antigen protein for treating a subject depends on the type of tumor being treated and the expression profile of the individual tumor. For example, a human suffering from prostate cancer is preferably overexpressed in a tumor antigen that is typically (or overexpressed) in prostate carcinoma, or specifically in a subject to be treated. It is treated with any of the tumor antigens, such as PSMA, PSCA, and / or PSA.

The coding sequence for the nucleic acid of the invention according to the first aspect of the invention comprises the coding sequence for a mono-, di-, or even multicistron nucleic acid, i.e., 1, 2, or more proteins or peptides. It may exist as a nucleic acid. Such coding sequences in di-or even multicistron nucleic acids can be, for example, by at least one internal ribosome entry site (IRES) sequence described herein, or by a resulting polypeptide comprising several proteins or peptides. It may be disrupted by a signal peptide that induces cleavage.

According to the first aspect of the present invention, the nucleic acid sequence of the present invention comprises a coding region encoding a peptide or protein containing a tumor antigen or a fragment, variant or derivative thereof. Preferably, the encoded tumor antigen is not a histone protein. In the present invention, such histone proteins are typically strongly alkaline proteins found in the nucleus of eukaryotic cells, directing DNA and packaging into structural units called nucleosomes. Histone proteins are the main protein components of chromatin, which act as spools of DNA around them and play a role in gene regulation. In the absence of histones, the DNA in the unwound chromosomes would be very long (in human DNA, the length-to-width ratio is more than 10 million to 1). For example, each human cell has about 1.8 meters of DNA, but wraps around histones to form about 90 millimeters of chromatin. Chromatin becomes a chromosome of approximately 120 micrometers when replicated and condensed during mitosis. More preferably, in the present invention, such histone proteins are typically selected from one of the following five major classes of histones: H1 / H5, H2A, H2B, H3, and H4 ", preferably. Is defined as a highly conserved protein selected from mammalian histones, more preferably from human histones or histone proteins. Such histones or histone proteins are typically histones H2A, H2B, H3, and. It is organized into two superclasses defined as core histones containing "H4" and linker histones containing histones H1 and H5.

In this regard, linker histones, preferably mammalian linker histones, more preferably human linker histones, which are preferably excluded from the scope of protection of the pending invention, typically contain H1 (specifically, H1F). It is selected from H1F0, H1FNT, H1FOO, H1FX), and H1H1 (specifically, including HIST1H1A, HIST1H1B, HIST1H1C, HIST1H1D, HIST1H1E, HIST1H1T).

Furthermore, core histones, preferably mammalian core histones, more preferably human core histones, which are preferably excluded from the scope of the pending invention, typically include H2AFs (specifically, H2AFB1, H2AFB2, H2AFB3, H2AFJ, H2AFV, H2AFX, H2AFY, H2AFY2, H2AFZ included), and H2A1 (specifically, HIST1H2AA, HIST1H2AB, HIST1H2AC, HIST1H2AD, HIST1H2AE, HIST1H2AG, HIST1H2AE, HIST1H2AG, HIST1H2AI H2A including HIST2H2AA3, HIST2H2AC; H2BF (specifically, H2BFM, H2BFO, H2BFS, H2BFWT), H2B1 (specifically, HIST1H2BA, HIST1H2BB, HIST1H2B, HIST1H2B, HIST1H2B , HIST1H2BI, HIST1H2BJ, HIST1H2BK, HIST1H2BL, HIST1H2BM, HIST1H2BN, HIST1H2BO, and H2B2 (specifically, including HIST2H2BO) , HIST1H3F, HIST1H3G, HIST1H3H, HIST1H3I, HIST1H3J), and H3A2 (specifically, including HIST2H3C), and H3A3 (specifically, including HIST3H3). HIST1H4A, HIST1H4B, HIST1H4C, HIST1H4D, HIST1H4E, HIST1H4F, HIST1H4G, HIST1H4H, HIST1H4I, HIST1H4J, HIST1H4I, HIST1H4J, HIST1H4K, HIST1H4K, HIST1H4K, HIST1H4K

According to the first aspect of the present invention, the nucleic acid sequence of the present invention comprises a coding region encoding a peptide or protein containing a tumor antigen or a fragment, variant or derivative thereof. Preferably, the encoded tumor antigen is not a reporter protein (eg, luciferase, green fluorescent protein (GFP), sensitive green fluorescent protein (EGFP), β-galactosidase) and is not a marker or selective protein (eg, alpha). Globin, galactokinase, and xanthin: not guanine phosphoribosyl transferase (GPT)). Preferably, the nucleic acid sequence of the present invention does not contain a (bacterial) antibiotic resistance gene, particularly a neo gene sequence (neomycin resistance gene) or a CAT gene sequence (chloramphenicol acetyltransferase; chloramphenicol resistance gene). ..

As defined above, the nucleic acids of the invention are a) a coding region encoding a peptide or protein containing a tumor antigen or fragment, variant, or derivative thereof, and (preferably said peptide or the encoded protein or protein. The peptide or protein comprising or encoding b) at least one histone stem loop and c) a poly (A) sequence or polyadenylation signal is preferably used to increase protein expression. , As defined above, are not histone proteins, reporter proteins, and / or markers or selective proteins. The nucleic acid elements b) to c) of the present invention may be present in the nucleic acid of the present invention in any order, that is, the elements a), b), and c) are 5'of the nucleic acid sequence of the present invention. It may exist in the order of a), b), and c) or a), c), and b) in the 3'direction from 5'-CAP structure, poly (C) sequence, stabilized sequence, IRES sequence. Etc., which may contain additional elements as defined herein. Each element a) to c) of the nucleic acid of the present invention, in particular a) and / or each element b) and c), more preferably element b) in a di- or multicistron construct, is at least in the nucleic acid of the present invention. It may be repeated once, preferably two or more times. As an example, the nucleic acids of the invention may contain sequence elements a), b), and optionally c), eg, in the following order:
5'-coding region-histon stem-loop-poly (A) sequence-3'; or 5'-coding region-histon stem-loop-polyadenylation signal-3'; or 5'-coding region-poly (A) sequence- Histon stem-loop-3'; or 5'-coding region-polyadenylation signal-histon stem-loop-3'; or 5'-coding region-coding region-histon stem-loop-polyadenylation signal-3'; or 5'- Code region-Histon stem loop-Histon stem loop-Poly (A) sequence-3'; or 5'-Code region-Histon stem loop-Histon stem loop-Polyadenylation signal-3'etc.

In this regard, the nucleic acid sequences of the invention are a) a coding region encoding a peptide or protein containing a tumor antigen or fragment, variant, or derivative thereof, and (preferably said above. The protein comprising or encoding b) at least one histone stem loop and c) a poly (A) sequence or a polyadenylated sequence is preferred to increase the expression level of the peptide or protein. Is a histone protein, a reporter protein (eg, luciferase, GFP, EGFP, β-galactosidase, especially EGFP), and / or a marker or selective protein (eg, alphaglobin, galactoxinase, and xanthin: guanine phosphoribosyl transferase (GPT)). )is not it.

In a more preferred embodiment of the first aspect, the nucleic acid sequence of the invention as defined herein may be present in the form of a modified nucleic acid.

In this regard, the nucleic acid sequences of the invention as defined herein are for "stabilized nucleic acid", preferably stabilized RNA, more preferably for degradation in vivo (eg, by exonucleases or endonucleases). It may be modified to provide RNA that is inherently resistant. Stabilized nucleic acids are introduced, for example, by introducing nucleotide analogs (eg, nucleotides with skeletal, sugar-modified, or base-modified) by varying the G / C content of the coding region of the nucleic acid sequence of the invention. Alternatively, it can be obtained by introducing a stabilizing sequence into the 3'and / or 5'untranslated region of the nucleic acid sequence of the present invention.

As described above, the nucleic acid sequences of the present invention as defined herein may contain nucleotide analogs / modifications such as skeletal modifications, sugar modifications, base modifications and the like. The skeletal modification related to the present invention is a modification in which the phosphoric acid of the skeleton of the nucleotide contained in the nucleic acid sequence of the present invention as defined herein is chemically modified. The sugar modification related to the present invention is a chemical modification of the sugar of the nucleotide of the nucleic acid sequence of the present invention as defined herein. Furthermore, the base modification related to the present invention is a chemical modification of the base portion of the nucleotide of the nucleic acid molecule of the nucleic acid sequence of the present invention. In this regard, nucleotide analogs or modifications are preferably selected from nucleotide analogs applicable to transcription and / or translation.

In a particularly preferred embodiment of the first aspect of the invention, the nucleotide analog / modification defined herein further increases the expression of the encoded protein and is preferably selected from base modifications selected from: 2-Amino-6-chloropurineriboside-5'-triphosphate, 2-aminoadenosine-5'-triphosphate, 2-thiocytidine-5'-triphosphate, 2-thiouridine-5'-triphosphate, 4-Thiouridine-5'-triphosphate, 5-aminoallylcytidine-5'-triphosphate, 5-aminoallyluridine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, 5-bromouridine- 5'-triphosphate, 5-iodocytidine-5'-triphosphate, 5-iodouridine-5'-triphosphate, 5-methylcytidine-5'-triphosphate, 5-methyluridine-5'-triphosphate, 6-azacytidine-5'-triphosphate, 6-azauridine-5'-triphosphate, 6-chloropurineriboside-5'-triphosphate, 7-deazaadenosine-5'-triphosphate, 7-deazaguanosine -5'-triphosphate, 8-azaadenosine-5'-triphosphate, 8-azidoadenosine-5'-triphosphate, benzimidazole-riboside-5'-triphosphate, N1-methyladenosine-5'-triphosphate , N1-methylguanosine-5'-triphosphate, N6-methyladenosine-5'-triphosphate, O6-methylguanosine-5'-triphosphate, pseudouridine-5'-triphosphate, or puromycin-5'-tri Phosphate, xanthosine-5'-triphosphate. From a group of base-modified nucleotides consisting of 5-methylcytidine-5'-triphosphate, 7-deazaguanosine-5'-triphosphate, 5-bromocytidine-5'-triphosphate, and pseudouridine-5'-triphosphate. Nucleotides for base modification of choice are particularly preferred.

According to a further embodiment, the nucleic acid sequences of the invention as defined herein may contain lipid modifications. Such lipid-modified nucleic acids typically include the nucleic acids defined herein. Such lipid-modified nucleic acid molecules of the nucleic acid sequences of the invention as defined herein typically have at least one linker covalently attached to said nucleic acid molecule and at least one covalently linked to each said linker. Further contains one lipid. Alternatively, the lipid-modified nucleic acid molecule comprises at least one nucleic acid molecule as defined herein and at least one (bifunctional) lipid covalently attached (without a linker) to the nucleic acid molecule. According to the third option, the lipid-modified nucleic acid molecule is covalently attached to the nucleic acid molecule as defined herein, at least one linker covalently attached to the nucleic acid molecule, and each of the linkers. It comprises at least one lipid and at least one (bifunctional) lipid that is covalently attached (without a linker) to the nucleic acid molecule. In this regard, it is particularly preferred that the lipid modification be present at the end of the linear nucleic acid sequence of the invention.

According to another preferred embodiment of the first aspect of the invention, the nucleic acid sequences of the invention as defined herein are the addition of a so-called "5'CAP" structure, especially when provided as (m) RNA. Can be stabilized against RNase degradation.

According to a more preferred embodiment of the first aspect of the invention, the nucleic acid sequences of the invention as defined herein are at least 10 cytidines, preferably at least 20 cytidines, more preferably at least 30. It may be modified by a sequence of cytidine (so-called "poly (C) sequence". Preferably, the nucleic acid sequence of the present invention typically comprises from about 10 to about 200 cytidine nucleotides, preferably from about 10 to about. Poly (C) sequence of 100 cytidine nucleotides, more preferably about 10 to about 70 cytidine nucleotides, or even more preferably about 20 to about 50, or even 20 to 30 cytidine nucleotides. The poly (C) sequence is preferably located at 3'of the coding region contained in the nucleic acid of the present invention according to the first aspect of the present invention. ..

In a particularly preferred embodiment of the invention, a coding region of the nucleic acid sequence of the invention as defined herein encoding at least one peptide or protein comprising a tumor antigen or fragment, variant or derivative thereof. The G / C content is varied, preferably increased, relative to the G / C content of that particular wild-type coding region, i.e., the unmodified coding region. The amino acid sequence encoded by the coding region is preferably unchanged compared to the amino acid sequence encoded by the particular wild-type coding region.

Changes in the G / C content of the coding region of the nucleic acid sequences of the invention as defined herein are based on the fact that the sequence of any mRNA region to be translated is important for efficient translation of that mRNA. There is. Therefore, the composition and sequence of various nucleotides is important. Specifically, mRNA sequences with high G (guanosine) / C (cytosine) content are more stable than mRNA sequences with high A (adenosine) / U (uracil) content. According to the present invention, the codons of the coding region are changed with respect to the wild-type coding region while retaining the amino acid sequence to be translated so that the amount of G / C nucleotides contained is increased. In connection with the fact that several codons encode one and the same amino acid (so-called degeneration of the genetic code), the most preferred codon for stability can be determined (so-called alternative codons). use).

Depending on the amino acid encoded by the coding region of the nucleic acid sequence of the present invention as defined herein, there is a possibility that the nucleic acid sequence, for example, the coding region can be variously modified with respect to the wild-type coding region. For amino acids encoded by codons containing only G or C nucleotides, the codons do not need to be modified. Therefore, the codons of Pro (CCC or CCG), Arg (CGC or CGG), Ala (GCC or GCG), and Gly (GGC or GGG) do not need to be modified because neither A nor U is present.

In contrast, codons containing A and / or U nucleotides may be modified by substituting with other codons that encode the same amino acid but do not contain A and / or U. An example is:
The codon of Pro may be modified from CCU or CCA to CCC or CCG;
The codon of Arg may be modified from CGU or CGA or AGA or AGG to CGC or CGG;
Ala codons may be modified from GCU or GCA to GCC or GCG;
The codon of Gly may be modified from GGU or GGA to GGC or GGG.

In other cases, the A or U nucleotides cannot be eliminated from the codons, but the A and U contents can be reduced by using codons with a low content of A and / or U nucleotides. An example is:
The Ph codon may be modified from UUU to UUC;
The Leu codon may be modified from UUA, UUG, CUU or CUA to CUC or CUG;
The codon of Ser may be modified from UCU or UCA or AGU to UCC, UCG or AGC;
The codon of Tyr may be modified from UAU to UAC;
The Cys codon may be modified from UGU to UGC;
The His codon may be modified from CAU to CAC;
The Gln codon may be modified from CAA to CAG;
The codon of Ile may be modified from AUU or AUA to AUC;
The codon of Thr may be modified from ACU or ACA to ACC or ACG;
The Asn codon may be modified from AAU to AAC;
The Lys codon may be modified from AAA to AAG;
The Val codon may be modified from GUU or GUA to GUC or GUG;
The Asp codon may be modified from GAU to GAC;
The codon of Glu may be modified from GAA to GAG;
The stop codon UAA may be modified to UAG or UGA.

On the other hand, in the case of Met (AUG) and Trp (UGG) codons, the sequence cannot be modified.

The substitutions individually or in whole to increase the G / C content of the coding region of the nucleic acid sequence of the invention as defined herein as compared to that particular wild-type coding region (ie, the original sequence). It can be used in any possible combination. Thus, for example, all codons of Thr present in the wild-type sequence may be modified to ACC (or ACG).

In the above, the codon of mRNA is shown. Therefore, the uridine present in the mRNA can be present as a thymidine in each DNA encoding a particular mRNA.

The G / C content of the coding region of the nucleic acid sequence of the present invention as defined herein is preferably at least 7%, more preferably at least 15%, particularly preferably at least 15% of the G / C content of the wild-type coding region. Increase by at least 20%. According to certain embodiments, at least 5%, 10% of the substitutable codons in the coding region encoding at least one peptide or protein, including the tumor antigen or fragments, variants, or derivatives thereof. Substituting 20%, 30%, 40%, 50%, 60%, more preferably at least 70%, even more preferably at least 80%, most preferably at least 90%, 95%, or even 100%. Increase the G / C content of the coding region.

In this regard, increasing the G / C content of the coding region of the nucleic acid sequence of the invention as defined herein to the maximum (ie, 100% of the substitutable codons) as compared to the wild-type coding region. Is particularly preferable.

According to the invention, a further preferred modification of the coding region of the nucleic acid sequence of the invention as defined herein encoding at least one peptide or protein, including a tumor antigen or fragment, variant or derivative thereof. Is based on the finding that the translation efficiency is also determined by the different frequency of occurrence of intracellular tRNA. Therefore, if a so-called "rare codon" is present in the coding region of the nucleic acid sequence of the invention as defined herein, the modified nucleic acid sequence corresponding to the rare codon encodes a relatively "frequent" tRNA. It is more likely to be translated significantly less than if the codons were present.

In this regard, the coding region of the nucleic acid sequence of the invention preferably has at least one codon of the wild-type sequence encoding a relatively rare tRNA in the cell relatively high in the cell. It is modified for the corresponding wild-type coding region so that it encodes a frequently present tRNA and is exchanged for a codon that carries the same amino acids as the relatively rare tRNA. By this modification, the coding region of the nucleic acid sequence of the present invention as defined herein is modified so that a codon that can utilize a frequently present tRNA is inserted. In other words, according to the present invention, with this modification, all codons in the wild-type coding region encoding a relatively rare tRNA in the cell are present in the cell at a relatively high frequency and in each case. May be exchanged for codons that carry the same amino acids as the relatively rare tRNA.

It is known to those skilled in the art which tRNAs are present relatively frequently in cells and, in contrast, which tRNAs are present relatively infrequently. For example, Akashi, Curr. Opin. Genet. Dev. 2001, 11 (6): 660-666. Codons that use the most frequently present tRNAs for a particular amino acid, such as Gly codons that use the most frequently present tRNAs in (human) cells, are particularly preferred.

According to the present invention, the G / C content of a sequence in the coding region of the nucleic acid sequence of the invention as defined herein without modifying the amino acid sequence of the peptide or protein encoded by the coding region of the nucleic acid sequence. Is particularly preferred to combine (especially maximally) with "frequent" codons. This preferred embodiment can provide the nucleic acid sequences of the invention as defined herein that are particularly efficiently translated and stabilized (modified).

According to another preferred embodiment of the first aspect of the invention, the nucleic acid sequence of the invention as defined herein preferably further comprises at least one 5'and / or 3'stabilized sequence. These stabilized sequences in the 5'and / or 3'untranslated regions have the effect of prolonging the half-life of mRNA in nucleic acids, especially cytosols. These stabilized sequences may have 100% sequence identity with natural sequences present in viruses, bacteria, and prokaryotes, but may be partially or completely synthesized. For example, an example of a stabilized sequence that can be used in the present invention for a stabilized nucleic acid is the untranslated sequence (UTR) of the (alpha) globin gene from human or Xenopus laevis. Another example of a stabilizing sequence is a sequence having the general formula (C / U) CCAN _x CCC (U / A) Py _x UC (C / U) CC (SEQ ID NO: 55), which is (alpha). It is contained in the 3'-UTR of a highly stable RNA encoding globin, type (I) collagen, 15-lipoxygenase, or tyrosine hydroxylase (Holcik et al., Proc. Natl. Acad. Sci. USA 1997, 94: 2410-2414). Of course, such stabilized sequences may be used individually or in combination with each other, or may be used in combination with other stabilized sequences known to those skilled in the art. In this regard, the 3'UTR sequence of the alpha globin gene is at least one containing a tumor antigen, or a fragment, variant, or derivative thereof, contained in the nucleic acid sequence of the present invention according to the first aspect of the present invention. It is particularly preferably located at 3'in the coding region encoding the peptide or protein.

Substitution, addition, or removal of bases is preferably defined herein using a DNA matrix for preparing nucleic acid sequences by well-known site-specific mutagenesis techniques or using oligonucleotide ligation strategies. (See, eg, Maniatis et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor Laboratory Press, 3rd ed., Cold Spring Harbor, N.). Any of the above modifications, when used in the present invention, can be applied to the nucleic acid sequences of the present invention as defined herein, as well as any nucleic acid, and if preferred or necessary, a combination of these modifications is the respective nucleic acid. Can be combined with each other in any combination as long as they do not interfere with each other. Those skilled in the art can make appropriate choices.

Nucleic acid sequences used according to the present invention as defined herein include, for example, in vitro methods such as in vitro transcription reactions, or in vivo reactions such as in vivo proliferation of DNA plasmids in bacteria, in addition to synthetic methods such as solid phase synthesis. It can be prepared using any method known in the art.

In such a process for preparing the nucleic acid sequences of the invention as defined herein, the corresponding DNA molecule may be transcribed in vitro, especially if the nucleic acid is in the form of mRNA. This DNA matrix preferably comprises a suitable promoter, such as the T7 or SP6 promoter, for in vitro transcription, followed by the desired nucleotide sequence of the nucleic acid molecule (eg, mRNA) prepared, and for in vitro transcription. The termination signal follows. The DNA molecules that form the matrix of at least one RNA of interest can be prepared by fermentation and proliferation and then isolation as part of a plasmid that can replicate in bacteria. Plasmids that may be suitable for the present invention include, for example, plasmid pT7T (Genbank Accession No. U26404; Lai et al., Development 1995, 121: 2349-2360), pGEM® series, eg, pGEM®. -1 (Genbank accession number X65300; manufactured by Promega), and pSP64 (Genbank accession number X65327). See also Griffin and Griffin (ed.), PCR Technology: Current Innovation, CRC Press, Boca Raton, FL, 2001 Mezi and Forts, Purification of PCR Products.

In addition to the nucleic acid sequences of the invention defined herein, the proteins or peptides encoded by this nucleic acid sequence may include fragments or variants of these sequences. Such fragments or variants are typically proteins or peptides at the nucleic acid or amino acid level, relative to the whole wild sequence, when encoded by one of the above nucleic acids, or by the nucleic acid sequences of the invention. Or at least 5%, 10%, 20%, 30%, 40%, 50%, 60%, preferably at least 70%, more preferably at least 80%, more preferably at least 85%, with one of the sequences. Even more preferably, it may comprise a sequence having at least 90%, most preferably at least 95%, or even 97%, 98% or 99% sequence identity.

A "fragment" of a protein or peptide in the present invention (eg, as encoded by the nucleic acid sequence of the invention as defined herein) may include the sequence of the protein or peptide as defined herein. The sequence is N-terminal, C-terminal, and / or within the sequence relative to the amino acid sequence (or encoding nucleic acid molecule) of the original (native) protein with respect to its amino acid sequence (or encoding nucleic acid molecule). Is disconnected / shortened. Therefore, such cleavage can occur at the amino acid level or at the corresponding nucleic acid level. Therefore, sequence identity with respect to said fragments as defined herein may preferably refer to all proteins or peptides as defined herein, or all (coding) nucleic acid molecules of such proteins or peptides. Similarly, a "fragment" of a nucleic acid in the present invention may include a sequence of nucleic acids as defined herein, said sequence with respect to the nucleic acid molecule as compared to the nucleic acid molecule of the original (native) nucleic acid molecule. '3' and / or the sequence is truncated / shortened. Therefore, sequence identity with respect to such fragments as defined herein may preferably refer to all nucleic acids as defined herein, and preferred sequence identity levels are typically specified herein. It's a street. Fragments have the same biological function or specific activity (eg, specific antigenicity or therapeuticity) as compared to full-length native peptides or proteins, or at least 50%, more preferably, of the activity of natural full-length proteins. Antigenicity by at least 70%, and even more preferably at least 90% (appropriate functional assays, eg, appropriate assay systems that measure immune responses such as expression and / or secretion of appropriate cytokines (as indicators of immune response). At least retains (measured in the assay to be assayed). Thus, in a preferred embodiment, the "fragment" is part of a full-length (naturally occurring) tumor antigenic protein that exerts tumor antigenicity against the immune system as specified herein.

A fragment of a protein or peptide herein (eg, as encoded by the nucleic acid sequence of the invention as defined herein) is a book having an amino acid length of about 6 amino acids to about 20 amino acids, or even longer. It may further comprise a sequence of a protein or peptide as defined herein, wherein the sequence is, for example, preferably about 8 amino acids long to about 10 amino acids long (eg, 8 amino acids long, 9 amino acids long, or 10 amino acids long). Fragments processed and presented by MHC class I molecules having (or even 6 amino acid length, 7 amino acid length, 11 amino acid length or 12 amino acid length)), or preferably about 13 or more amino acid lengths (eg, 13). Treated and presented by an MHC class II molecule having amino acid length, 14 amino acid length, 15 amino acid length, 16 amino acid length, 17 amino acid length, 18 amino acid length, 19 amino acid length, 20 amino acid length, or even longer amino acid length). These fragments may be selected from any part of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of peptide fragments and MHC molecules, i.e., said fragments are typically not recognized in their native form. Fragments of proteins or peptides as defined herein may contain at least one epitope of these proteins or peptides. Further, protein domains such as extracellular domains, intracellular domains, or transmembrane domains, and shortened or truncated forms of proteins may also be understood to include fragments of the protein.

Also, fragments of proteins or peptides as defined herein (eg, as encoded by the nucleic acid sequences of the invention as defined herein) may comprise epitopes of these proteins or peptides. In the present invention, a part of the T cell epitope or protein may preferably further contain a fragment having an amino acid length of about 6 to about 20 amino acids, or even longer, for example, the fragment is preferably. Treated and presented by MHC class I molecules having a length of about 8 amino acids to about 10 amino acids (eg, 8 amino acids, 9 or 10 amino acids (or even 11 or 12 amino acids)). Fragments, or preferably about 13 or more amino acid lengths (eg, 13 amino acid length, 14 amino acid length, 15 amino acid length, 16 amino acid length, 17 amino acid length, 18 amino acid length, 19 amino acid length, 20 amino acid length, or even more. Fragments processed and presented by MHC class II molecules having (longer amino acid lengths), these fragments may be selected from any portion of the amino acid sequence. These fragments are typically recognized by T cells in the form of a complex consisting of peptide fragments and MHC molecules, i.e., said fragments are typically not recognized in their native form.
B-cell epitopes typically have a (native) protein or peptide antigen as defined herein having preferably 5 amino acids to 15 amino acids, more preferably 5 amino acids to 12 amino acids, even more preferably 6 amino acids to 9 amino acids. A fragment located on the outer surface of an amino acid, which can be recognized by its native form of the amino acid.
Such epitopes of a protein or peptide may further be selected from any of the variants mentioned herein of such proteins or peptides. In this regard, the epitope determinants are discontinuous in the amino acid sequence of the protein or peptide defined herein, but are composed of adjacent segments of the protein or peptide defined herein in a three-dimensional structure. It may be a higher-order structural epitope or a discontinuous epitope, or it may be a continuous epitope or a linear epitope composed of a single polypeptide chain.

A "variant" of a protein or peptide as defined in the present invention can be encoded by the nucleic acid sequence of the present invention as defined herein. Thereby, an amino acid sequence different from the original sequence due to one or more (2, 3, 4, 5, 6, 7, or more) mutations such as substitution, insertion, and / or deletion of one or more amino acids can be obtained. The protein or peptide it has can be produced. The preferred sequence identity level of a "mutant" in a full-length intrinsically disordered protein sequence is typically as specified herein. Preferably, the variant has the same biological function or specific activity (eg, specific antigenicity) or at least 50% of the activity of the native full-length protein, more preferably compared to the full-length native peptide or protein. Holds at least 70%, and even more preferably at least 90% (measured by an appropriate functional assay, eg, an assay that assayes the immune response to a tumor antigen by secretion and / or expression of one or more cytokines). Thus, in a preferred embodiment, a "mutant" is a variant of a tumor antigen protein that exhibits tumor antigenicity to the extent specified herein.

A "variant" of a protein or peptide as defined in the present invention (eg, as encoded by a nucleic acid as defined herein) is compared to a native, i.e., non-mutated physiological sequence. , May include conservative amino acid substitutions. In particular, in addition to these amino acid sequences, the nucleotide sequences encoding them are also included in the term variant as defined herein. Substitutions in which amino acids from the same classification are exchanged are called conservative substitutions. Specifically, an aliphatic side chain, a positively or negatively charged side chain, an aromatic group in a side chain or an amino acid, a side chain capable of hydrogen bonding, for example, an amino acid having a side chain having a hydroxyl functional group be. The conservative substitution is, for example, that an amino acid having a polar side chain is replaced by another amino acid that also has a polar side chain, or, for example, an amino acid characterized by a hydrophobic side chain has a hydrophobic side chain as well. It means that it is replaced by another amino acid that it has (for example, serine (threonine) is replaced by threonine (serine), or leucine (isoleucine) is replaced by isoleucine (leucine)). In particular, insertions and substitutions are possible at sequence positions that do not change the three-dimensional structure or affect the binding region. Changes in three-dimensional structure due to insertion or deletion can be easily determined using, for example, a CD spectrum (circular dichroism spectrum) (Modern Physical Methods in Biochemistry, Newberger et al. (Ed.), Elsevier. , Urry in Amsterdam, 1985, Absorption, Circular Dichroism and ORD of Polypeptides).

Furthermore, in the proteins or peptide variants defined herein that can be encoded by the nucleic acid sequences of the invention as defined herein, the nucleotides of the nucleic acids do not alter the respective amino acid sequences of the proteins or peptides. May contain sequences that have been exchanged according to the degeneracy of the genetic code, i.e., the amino acid sequence or at least a portion thereof, in the above sense, may contain one or more mutations to be an original sequence. It may not be different.

To determine the proportion of two sequences, eg, the nucleic acid sequence or amino acid sequence defined herein, preferably the amino acid sequence or amino acid sequence itself encoded by the nucleic acid sequence of the invention defined herein. In addition, the sequences may be aligned for later comparison with each other. This allows, for example, a position in the first sequence to be compared to a corresponding position in the second sequence. If a position in the first sequence is occupied by the same components as the corresponding position in the second sequence, the two sequences are identical at that position. If this is not the case, the sequences are different at that position. If the second sequence is inserted as compared to the first sequence, a gap may be inserted in the first sequence for further alignment. If the second sequence is missing compared to the first sequence, a gap may be inserted in the second sequence for further alignment. The ratio of two sequences being identical is then a function of the number of identical positions divided by the total number of positions including positions occupied in only one sequence. The proportion of two sequences that are identical can be determined using a mathematical algorithm. Preferred but non-limiting examples of mathematical algorithms that can be used are described in Karlin et al. (1993), PNAS USA, 90: 5873-5877 or Altschul et al. (1997), Nucleic Acids Res. , 25: 3389-3402. Such an algorithm is integrated into the BLAST program. Sequences that are to some extent identical to the nucleic acids of the invention can be identified by this program.

The nucleic acid sequences of the invention as defined herein can encode peptides or derivatives of proteins. Such a derivative of a peptide or protein is a molecule derived from another molecule such as the peptide or protein. A "derivative" typically comprises a full-length sequence of a native peptide or protein, with additional sequence features, eg, at the N-terminus or C-terminus, such sequence features relative to the native full-length peptide / protein. It can exert further functions. Also, such derivatives have the same biological function or specific activity (eg, specific therapeuticity) as a full-length native peptide or protein, or at least 50%, more preferably, of the activity of a natural full-length protein. It retains at least 70%, and even more preferably at least 90% (measured by a suitable functional assay (see above), eg, as represented by expression and / or secretion of cytokines in an immune response). Thus, a "derivative" of a peptide or protein may be, for example, a (chimeric) fusion peptide / protein containing the peptide or protein used in the present invention, or another peptide / protein that imparts two or more biological functions to the fusion peptide / protein. It also includes a natural full-length protein (or variant / fragment thereof) fused with. For example, the fusion comprises a label, such as an epitope such as a FLAG epitope or a V5 epitope or an HA epitope. For example, the epitope is a FLAG epitope. Such tags are useful, for example, in the purification of fusion proteins.

In this regard, a "mutant" of a protein or peptide is at least 70%, 75 over an amino acid extension of 10, 20, 30, 50, 75, or 100 of the protein or peptide. It can have%, 80%, 85%, 90%, 95%, 98% or 99% amino acid identity. Similarly, a "mutant", or in particular a "fragment" of a nucleic acid sequence, is at least 70% over an extension of 10, 20, 30, 50, 75, or 100 nucleotides of the nucleic acid sequence. It can have 75%, 80%, 85%, 90%, 95%, 98% or 99% nucleotide identity, but typically refers to naturally occurring full-length sequences. In the case of a "fragment," sequence identity for a fragment typically extends over the length (of the fragment) in the portion of the full-length protein that exhibits the highest level of sequence identity (representing the same length as the fragment). It is determined.

In a more preferred embodiment of the first aspect of the invention, the nucleic acid sequence of the invention is vehicle, transfecting agent, or complex in order to increase the transfection efficiency and / or immunostimulation of the nucleic acid sequence of the invention. Combine with the agent. Particularly preferred agents suitable for increasing transfection efficiency in this regard are cationic or polycationic compounds, such as protamine, nucleolin, spermin or spermidin, or other cationic peptides or proteins. (For example, poly-L-lysine (PLL), polyarginine, basic polypeptide, cell permeable peptide (CPP) (HIV binding peptide, HIV-1 Tat (HIV), Tat-derived peptide, penetratin, VP22-derived peptide or (Including analog peptides), HSV VP22 (simple herpes), MAP, KALA, or protein transfection domain (PTD), PpT620, proline-rich peptide, arginine-rich peptide, lysine-rich peptide, MPG-peptide, Pep-1, L- Oligomers, calcitonin peptides, antennapedia-derived peptides (especially from Drosophila antennapedia), pAntp, pIsl, FGF, lactoferrin, transportan, buforin-2, Bac715-24, SynB, SynB (1), pVEC, hCT-derived peptides, SAP, or histone). Further, preferred cationic or polycationic proteins or peptides may be selected from proteins or peptides having the following formulas: (Arg) _l ; (Lys) _m ; (His) _n ; (Orn) _o ; Xaa) _x (in the formula, l + m + n + o + x = 8 to 15, and l, m, n, or o are independently of each other, 0, 1, 2, 3, 4, 5, 6, 7, 8, 9, It may be any number selected from 10, 11, 12, 13, 14 or 15, provided that the total content of Arg, Lys, His and Orn is at least 50% of the total amino acids of the oligopeptide; Xaa. Can be any amino acid selected from native (= naturally occurring) or non-native amino acids except Arg, Lys, His or Orn; x is selected from 0, 1, 2, 3 or 4 The total content of Xaa does not exceed 50% of the total amino acids of the oligopeptide). In this regard, particularly preferred cationic peptides are, for example, Arg ₇ , Arg ₈ , Arg ₉ , H ₃ R ₉ , R ₉ H ₃ , H ₃ R ₉ H ₃ , YSSR ₉ SSY, (RKH) ₄ , Y (RKH) ₂ R and the like. More preferred cationic or polycationic compounds that can be used as transfectants are cationic polysaccharides (eg, chitosan), polybrene, cationic polymers (eg, polyethyleneimine (PEI)), cationic lipids (eg, eg, polyethyleneimine). , DOTMA: [1- (2,3-thioleyloxy) propyl)]-N, N, N-trimethylammonium chloride, DMRIE, di-C14-amidine, DOTIM, SAINT, DC-Chol, BGTC, CTAP, DOPC , DODAP, DOPE: Dioleyl phosphatidylethanolamine, DOSPA, DODAB, DOIC, DMEPC, DOGS: Dioctadecylamide glycylspermine, DIMRI: Dimilist-oxypropyldimethylhydroxyethylammonium bromide, DOTAP: Dioreoil Oxy-3- ( Trimethylammonio) Propane, DC-6-14: O, O-ditetradecanoyl-N- (α-trimethylammonioacetyl) diethanolamine chloride, CLIP1: rac-[(2,3-dioctadecyloxypropyl) ( 2-Hydroxyethyl)]-dimethylammonium chloride, CLIP6: rac- [2 (2,3-dihexadecyloxypropyl-oxymethyloxy) ethyl] trimethylammine, CLIP9: rac- [2 (2,3-dihexa) Decyloxypropyl-oxysuccinyloxy) ethyl] -trimethylammonium, oligofectamine), or modified polyamino acids such as cationic or polycationic polymers (eg, β-amino acid polymers or reverse polyamides; PVP (poly (N) -Modified polyethylene such as -ethyl-4-vinylpyridinium bromide); modified acrylate such as pDMAEMA (poly (dimethylaminoethylmethylmethacrylate)); modified amidamine such as pAMAM (poly (amideamine)); diamine-terminated modified 1,4 butane Modified polybetaaminoester (PBAE) such as diol diacrylate-co-5-amino-1-pentanol polymer; dendrimer such as polypropylamine dendrimer or pAMAM-based dendrimer; PEI: poly (ethyleneimine), poly (propyleneimine) Polyimine such as; polyallylamine; cyclodextrine-based polymer, dextran-based polymer, glycoskeletal polymer such as chitosan; PMOXA-PDMS Copo A silane skeleton polymer such as a limmer), a block polymer consisting of a combination of one or more cationic blocks (eg, selected from the cationic polymers described above) and one or more hydrophilic or hydrophobic blocks (eg, polyethylene). Glycol) and the like may be contained.

In this regard, it is particularly preferred that the nucleic acids of the invention are at least partially complexed with a cationic or polycationic compound, preferably a cationic protein or peptide. Partially means that only part of the nucleic acid sequence of the invention is complexed with a cationic or polycationic compound, and the rest of the nucleic acid sequence of the invention is in uncomplexed (“free”) form. It means that there is. Preferably, the ratio of the complexed nucleic acid to the free nucleic acid is from about 5: 1 (w / w) to about 1:10 (w / w), more preferably from about 4: 1 (w / w) to about 1: 1. 8 (w / w), even more preferably selected from the range of about 3: 1 (w / w) to about 1: 5 (w / w) or 1: 3 (w / w), most preferably complex. The ratio of the embodied nucleic acid to the free nucleic acid is selected from a ratio of about 1: 1 (w / w).

The nucleic acid sequences of the invention are provided in naked form or are complexed, for example, with polycationic compounds of any chemical structure, preferably with polycationic (poly) peptides or synthetic polycationic compounds. It is preferably provided at. Preferably, the nucleic acid sequence is not provided with the packaging cells.

In a further aspect, the invention comprises a plurality of, ie, more than 1, preferably 2-10, more preferably 2-5, most preferably 2-4 nucleic acid sequences of the invention as defined herein. Provide objects or kits or parts of kits. The compositions of the invention preferably contain more than one nucleic acid sequence of the invention, preferably encoding various peptides or proteins, including various tumor antigens or fragments, variants, or derivatives thereof.

In a preferred embodiment, a plurality of (typically more than one, more than two, more than three, more than four, more than five, more than six, especially for use in the treatment of prostate cancer (PCa). Or at least a composition, kit or part of a kit of the invention comprising a nucleic acid sequence of the invention (meaning more than 10 nucleic acids, eg, 2-10, preferably 2-5). Including:
a) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen PSA or a fragment, variant, or derivative thereof.
b) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen PSMA or a fragment, variant, or derivative thereof.
c) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains the tumor antigen PSCA or a fragment, variant, or derivative thereof.
d) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains the tumor antigen STEAP-1 or a fragment, variant, or derivative thereof.

In a more preferred embodiment, multiple (typically more than one, more than two, more than three, more than four, more than five, six), especially for use in the treatment of non-small lung cancer (NSCLC). Compositions, kits or parts of kits of the invention comprising the nucleic acid sequences of the present invention (meaning more or more than 10 nucleic acids, eg, 2-10, preferably 2-5) nucleic acids. , At least include:
a) Nucleic acid sequence
i. A coding region encoding at least one peptide or protein, including the tumor antigen NY-ESO-1, or a fragment, variant, or derivative thereof.
ii. At least one histone stemloop, and iii. With a nucleic acid sequence containing or encoding a poly (A) sequence or a polyadenylation signal,
b) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen 5T4 or a fragment, variant, or derivative thereof.
c) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen survivin or a fragment, variant, or derivative thereof.

In yet another embodiment, multiple (typically more than one, more than two, more than three, more than four, more than five, six), especially for use in the treatment of non-small lung cancer (NSCLC). Compositions, kits or parts of kits of the invention comprising the nucleic acid sequences of more than 10 or more than 10 nucleic acids, eg, 2-10, preferably 2-5) nucleic acids of the invention. Includes at least:
a) Nucleic acid sequence
i. A coding region encoding at least one peptide or protein, including the tumor antigen NY-ESO-1, or a fragment, variant, or derivative thereof.
ii. At least one histone stemloop, and iii. With a nucleic acid sequence containing or encoding a poly (A) sequence or a polyadenylation signal,
b) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen 5T4 or a fragment, variant, or derivative thereof.
c) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains a tumor antigen survivin or a fragment, variant, or derivative thereof.
d) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein is a nucleic acid containing the tumor antigen MAGE-C1 or a fragment, variant, or derivative thereof. ,
e) A nucleic acid of the invention encoding at least one peptide or protein, wherein the encoded peptide or protein contains the tumor antigen MAGE-C2 or a fragment, variant, or derivative thereof.

According to a further aspect, the invention is described, for example: a) Nucleic acid sequences or plurals of the invention as defined herein, which are typically more than one, more than two, more than three, more than four. The nucleic acid sequences of the present invention as defined herein for more than 5, 6, or more than 10 nucleic acids (meaning, for example, 2 to 10 nucleic acids, preferably 2 to 5 nucleic acids). A step of providing a composition of the present invention containing the composition of the present invention, and b) a composition of the present invention containing the nucleic acid sequence of the present invention defined in the present specification or a plurality of nucleic acid sequences of the present invention defined in the present specification. Provided is a method for increasing the expression of a encoded peptide or protein, including, for example, a cell-free expression system, a step of applying or administering to a cell (eg, expression host cell or somatic cell), tissue, or organism. .. The method can be applied for experimental, research, diagnostic, commercial peptide or protein products, and / or therapeutic purposes. In this regard, compositions of the invention typically comprising the nucleic acid sequences of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein are typically prepared. Specifically, for example, in the naked or complex form, or as the pharmaceutical composition or vaccine described herein, preferably via transfection, or any of the administration methods described herein. By use, it is applied or administered to a cell-free expression system, cells (eg, expression host cells or somatic cells), tissues, or organisms. The method may be performed in vitro, in vivo, or ex vivo. The method may further be used in the treatment of a particular disease, particularly preferably in the treatment of cancer or tumor disease, as defined herein.

In this context, in vitro refers to a composition of the invention comprising a nucleic acid sequence of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein in an in vitro culture. Transfection or transduction into cells is defined herein. In vivo means that by applying the nucleic acid of the present invention or the composition of the present invention to an organism or an individual, the nucleic acid sequence of the present invention or the composition of the present invention containing a plurality of nucleic acid sequences of the present invention is applied to cells. Transfection or transduction is defined herein. Exobibo is a nucleic acid sequence or plural of the present invention, typically more than one, more than two, more than three, more than four, more than five, more than six, or ten, outside the organism or individual. Cells are transfected or transduced with a composition of the invention comprising a nucleic acid sequence of the invention (meaning more than one nucleic acid, eg, 2-10, preferably 2-5). , As defined herein to apply the transfected cells to said organism or individual.

Similarly, according to another aspect, the invention also comprises, for example, a nucleic acid sequence of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein. , For example, by applying or administering to a cell-free expression system, cells (eg, expression host cells or somatic cells), tissues, or organisms, preferably for diagnostic or therapeutic purposes. Provided are the use of a composition of the invention comprising a nucleic acid sequence of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein to increase expression of a protein. The use can be applied for experimental, research, diagnostic, commercial products of peptides or proteins, and / or therapeutic purposes. In this regard, compositions of the invention typically comprising the nucleic acid sequences of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein are typically prepared. Specifically, for example, in the naked or complex form, or as the pharmaceutical composition or vaccine described herein, preferably via transfection, or any of the administration methods described herein. By use, it is applied or administered to a cell-free expression system, cells (eg, expression host cells or somatic cells), tissues, or organisms. The use may be performed in vitro, in vivo, or ex vivo. The use may further be used in the treatment of a particular disease, particularly preferably in the treatment of cancer or tumor disease, as defined herein.

In yet another aspect, the invention also relates to an expression system of the invention comprising the nucleic acid sequence, or expression vector, or plasmid of the invention according to the first aspect of the invention. In this regard, the expression system is a cell-free expression system (eg, in vitro transcription / translation system), a cell expression system (eg, CHO) used for the expression of a peptide or protein (eg, an animal such as a plant or bovine). It may be a mammalian cell such as a cell, an insect cell, a yeast cell, a bacterial cell such as Escherichia coli), or an organism.

Yet another aspect of the invention is also preferably in naked or complex form, or as a pharmaceutical composition or vaccine as described herein, more preferably administered as described herein. Using any of the methods, a cell (eg, an expression host cell or body) comprises a composition of the invention comprising a nucleic acid sequence of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein. A nucleic acid that increases the expression of a peptide or protein that is encoded or administered to a cell), tissue, or organism, preferably as defined herein (eg, to treat a cancer or tumor disease). It relates to the use of a composition of the invention comprising a nucleic acid sequence of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein for preparing a composition.

Thus, in a particularly preferred embodiment, the invention is also optionally pharmaceutically with a composition of the invention comprising the nucleic acid of the invention as defined herein or a plurality of nucleic acid sequences of the invention as defined herein. Provided is a pharmaceutical composition comprising an acceptable carrier and / or vehicle.

As a first component, the pharmaceutical composition of the invention comprises at least one nucleic acid of the invention as defined herein.

As a second ingredient, the pharmaceutical composition of the present invention may optionally include at least one additional pharmaceutically active ingredient. In this regard, a pharmaceutically active ingredient is a compound having a therapeutic effect that cures, ameliorates, or prevents a particular indication or disease, preferably cancer or tumor disease, referred to herein. The compounds include, but are not limited to, peptides or proteins (preferably as defined herein), nucleic acids (preferably as defined herein), low molecular weight organics (therapeutically active). Compounds or inorganic compounds (molecular weight: less than 5,000, preferably less than 1,000), saccharides, antigens or antibodies (preferably as defined herein), therapeutic agents already known in related arts, antigen cells, Antigen cell fragments, cell fractions, cell wall components (eg, polysaccharides), modified, attenuated, or inactivated pathogens (eg, chemically or by irradiation) (viruses, bacteria, etc.), (preferably books). Examples thereof include adjuvants (as defined in the specification).

In addition, the pharmaceutical compositions of the present invention may comprise a pharmaceutically acceptable carrier and / or vehicle. In the present invention, the pharmaceutically acceptable carrier typically comprises a liquid or non-liquid base of the pharmaceutical composition of the present invention. When the pharmaceutical composition of the present invention is provided in liquid form, the carrier is typically pyrogen-free water; isotonic saline or a buffered (water) solution, such as a buffered solution of phosphoric acid, citric acid or the like. Is. The injection buffer may be hypertonic, isotonic, or hypotonic with respect to the particular reference medium. That is, the buffer may have a higher, identical, or lower salt content for a particular reference medium, where preferably at a concentration that does not damage cells by osmotic pressure or other concentration effects. The above-mentioned salt may be used. The reference medium can be used as a reference medium in a liquid used in an "in vivo" method, such as, for example, blood, lymph, cytosolic fluid, or other body fluid, or in an "in vitro" method such as a general buffer or liquid. It is a liquid that can be produced. Such general buffers or liquids are known to those of skill in the art. Ringer's lactate is particularly preferred as the liquid base.

However, for the pharmaceutical compositions of the present invention, one or more compatible solid or liquid fillers or diluents, or encapsulating compounds suitable for administration to the patient to be treated may be used as well. .. The term "compatible" as used herein is used in the present invention so that, under typical conditions of use, there is no interaction that substantially reduces the pharmaceutical efficacy of the pharmaceutical compositions of the present invention. It means that these components of the pharmaceutical composition can be mixed with the nucleic acid of the present invention as defined herein.

According to certain embodiments, the pharmaceutical composition of the present invention may comprise an adjuvant. In this regard, an adjuvant can be understood as any compound suitable for initiating or increasing the immune response of the innate immune system, i.e., a non-specific immune response. In other words, when administered, the pharmaceutical compositions of the invention preferably elicit an innate immune response with an optionally contained adjuvant. Preferably, such adjuvants are known to those of skill in the art and are suitable for the present case, i.e., select from adjuvants that support the induction of the innate immune response in mammals, such as the adjuvant proteins defined above or the adjuvants defined below. You can do it.

Particularly preferred as depots and adjuvants suitable for delivery are the cationic or polycationic compounds defined above for the nucleic acid sequences of the invention as vehicles, transfections, or complexing agents.

The pharmaceutical composition of the present invention may further contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory ability, if necessary. It is preferable that this provides a synergistic effect between the nucleic acid sequence of the present invention as defined herein and optionally an auxiliary substance that can be contained in the pharmaceutical composition of the present invention. In this regard, different mechanisms can be considered depending on the different types of auxiliary substances. For example, compounds that mature dendritic cells (DCs) (eg, lipopolysaccharides, TNF-alpha, or CD40 ligands) form a first class of suitable auxiliary substances. In general, any agent that affects the immune system, such as "danger signals" (LPS, GP96, etc.), or GM-CFS, etc. that can specifically enhance and / or affect the immune response. Cytokines can be used as an adjunct. Particularly preferred auxiliary substances are cytokines such as monokines, phosphokines, interleukins, or chemokines that further promote the natural immune response, such as IL-1, IL-2, IL-3, IL-4, IL-5, IL-. 6, IL-7, IL-8, IL-9, IL-10, IL-12, IL-13, IL-14, IL-15, IL-16, IL-17, IL-18, IL-19, IL-20, IL-21, IL-22, IL-23, IL-24, IL-25, IL-26, IL-27, IL-28, IL-29, IL-30, IL-31, IL- 32, IL-33, IFN-alpha, IFN-beta, IFN-gamma, GM-CSF, G-CSF, M-CSF, LT-beta, or growth factors such as TNF-alpha, hGH.

Further additives that may be included in the pharmaceutical compositions of the present invention are, for example, emulsifiers such as Tween®; wetting agents such as sodium lauryl sulfate; colorants; flavoring agents; pharmaceutical carriers; tablets; Stabilizer; antioxidant; preservative.

Also, the pharmaceutical compositions of the present invention are due to their binding affinity (as a ligand) for human Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, or mice. Known to be immunostimulatory due to binding affinity (as a ligand) for Toll-like receptors TLR1, TLR2, TLR3, TLR4, TLR5, TLR6, TLR7, TLR8, TLR9, TLR10, TLR11, TLR12 or TLR13. It may further contain any of the additional compounds listed above.

The pharmaceutical compositions of the present invention can be administered orally, parenterally, by inhalation spray, locally, intrarectally, nasally, intraorally, intravaginally, or via an implanted leather bag. May be administered. As used herein, the term parenteral is used subcutaneously, intravenously, intramuscularly, intraarterial, intrabursa, intramatrix, intrasheath, intrahepatic, intralesional, intracranial, transdermal, intradermal. Includes intrapulmonary, intraperitoneal, intracarpal, intraarterial, and sublingual injection or infusion techniques.

Preferably, the pharmaceutical composition of the present invention may be administered by parenteral injection, more preferably subcutaneously, intravenously, intramuscularly, intra-arterial, intrabursa, matrix, intrasheath, intrahepatic, lesions. It may be administered by injection or injection technique intracranial, intracranial, transdermal, intradermal, intrapulmonary, intraperitoneal, intracarpal, intraarterial, and sublingual. Intramuscular and intramuscular injections are particularly preferred. The sterile injectable form of the pharmaceutical composition of the present invention may be an aqueous or oily suspension. These suspensions can be formulated according to techniques known to those of skill in the art using suitable dispersants or wetting agents and suspending agents.

In addition, the pharmaceutical composition of the present invention as defined herein is orally administered in any orally acceptable dosage form including, but not limited to, capsules, tablets, aqueous suspensions, or solutions. good.

Also, the pharmaceutical compositions of the present invention are topical, especially if the therapeutic target comprises a region or organ readily accessible by topical application, including, for example, diseases of the skin or any other accessible epithelial tissue. It may be administered. Suitable topical formulations are readily prepared for each of these regions or organs. For topical application, the pharmaceutical composition of the invention may be formulated in a suitable ointment containing the nucleic acid of the invention as defined herein suspended or dissolved in one or more carriers.

The pharmaceutical compositions of the invention typically include "safe and effective amounts" of the components of the pharmaceutical composition of the invention, in particular the nucleic acids of the invention as defined herein. As used herein, a "safe and effective amount" is a nucleic acid sequence of the invention as defined herein that is sufficient to significantly induce a favorable change in the disease or illness as defined herein. Means the amount of. But at the same time, a "safe and effective amount" is an amount small enough to avoid serious side effects and to enable a reasonable relationship between benefits and risks. These limits are typically determined within reasonable medical judgment.

The pharmaceutical compositions of the present invention may be widely used as pharmaceutical compositions or vaccines in humans and for veterinary purposes, preferably for human medical purposes.

According to another particularly preferred embodiment, the pharmaceutical composition of the present invention (or the composition of the present invention comprising the nucleic acid sequence of the present invention as defined herein or a plurality of the nucleic acid sequences of the present invention as defined herein). May be provided or used as a vaccine. Typically, such vaccines are as defined above for the pharmaceutical composition. In addition, such vaccines typically contain a nucleic acid sequence of the invention as defined herein, or a composition of the invention comprising a plurality of nucleic acid sequences of the invention as defined herein.

Vaccines of the invention may also contain pharmaceutically acceptable carriers, adjuvants, and / or vehicles as defined herein for the pharmaceutical compositions of the invention. The choice of pharmaceutically acceptable carrier, particularly in relation to the vaccine of the invention, is, in principle, determined by the method of administering the vaccine of the invention. The vaccine of the present invention may be administered systemically or topically, for example. In general, systemic routes of administration include, for example, transdermal, oral, parenteral routes (including subcutaneous, intravenous, intramuscular, intra-articular, intradermal, and intraperitoneal injections, and / or intranasal routes of administration). Can be mentioned. In general, local routes of administration include, for example, intradermal, transdermal, subcutaneous, or intramuscular injections, or intralesional, intracranial, intrapulmonary, intracarpal, and sublingual. Injections are also included. More preferably, the vaccine may be administered by the transdermal, subcutaneous or intramuscular route. Therefore, the vaccines of the invention are preferably formulated in liquid (or sometimes solid) form.

The vaccine of the present invention may further contain one or more auxiliary substances in order to increase its immunogenicity or immunostimulatory ability, if necessary. An adjuvant as an adjunct or additive as defined above for the pharmaceutical composition is particularly preferred.

The present invention further comprises the present invention of a pharmaceutical composition of the present invention, a composition of the present invention comprising a plurality of the nucleic acid sequences of the present invention defined herein, of the nucleic acid sequences of the present invention defined herein. Provided are some uses and uses of the vaccines of the invention, all of which include the nucleic acid sequences of the invention as defined herein, or kits containing them.

According to one particular aspect, the invention is a composition of the invention comprising a plurality of the nucleic acid sequences of the invention as defined herein, particularly cancer. Alternatively, it relates to a first pharmaceutical use as a medicine in the treatment of a tumor disease, preferably as a vaccine.

According to another aspect, the invention is the present invention of the nucleic acid sequence of the invention as defined herein, or a plurality of the inventions as defined herein, for the treatment of cancer or tumor disease as defined herein. The present specification for preparing a second pharmaceutical use of the composition of the invention comprising the nucleic acid sequence of the invention, preferably a medicament for preventing, treating, and / or ameliorating a cancer or tumor disease as defined herein. Concerning the use of a composition of the present invention containing a plurality of the nucleic acid sequences of the present invention defined in the present invention, a pharmaceutical composition or a vaccine containing them, or a kit containing them. .. Preferably, the pharmaceutical composition or vaccine is used or administered to a patient in need of it for this purpose.

Preferably, the diseases referred to herein are selected from cancer or tumor diseases, wherein the cancer or tumor diseases include, for example, preferably: acute lymphoblastic leukemia, acute myeloid leukemia,. Adrenal cortex cancer, AIDS-related cancer, AIDS-related lymphoma, anal cancer, worm drop cancer, stellate cell tumor, basal cell cancer, total bile duct cancer, bladder cancer, bone cancer, osteosarcoma / malignant fibrous histiocytoma, brain stem glioma Tumors, brain tumors, cerebral stellate cell tumors, cerebral stellate cell tumors / malignant gliomas, ventricular lining cell tumors, medulloblastomas, primordial primordial neuroembroidered tumors above the tent, tract and hypothalamic gliomas, Cancer, bronchial adenomas / cartinoids, Berkit lymphoma, juvenile cartinoids, gastrointestinal cartinoids, cancers of unknown primary origin, primary central nervous system lymphoma, juvenile cerebral stellate collagenoma, juvenile cerebral stellate collagen / malignant nerve Glio, cervical cancer, childhood cancer, chronic lymphocytic leukemia, chronic myeloid leukemia, chronic myeloproliferative disease, colon cancer, cutaneous T-cell lymphoma, fibrogenic small round cell tumor, endometrial cancer, ventricular coat Celloma, esophageal cancer, Ewing sarcoma in Ewing tumor, juvenile extracranial embryonic cell tumor, extragonal embryonic cell tumor, extrahepatic bile duct cancer, intraocular melanoma, retinoblastoma, bile sac cancer, gastric cancer, gastrointestinal cartinoid, Gastrointestinal stromal tumor (GIST), extracranial, extragonadal, or ovarian germ cell tumor, gestational chorionic villus tumor, brain stem glioma, juvenile cerebral stellate cell tumor, juvenile tract and hypothalamic glioma , Gastric cartinoid, hairy cell leukemia, head and neck cancer, heart cancer, hepatocellular (liver) cancer, hodgkin lymphoma, hypopharyngeal cancer, juvenile hypothalamus and tract glioma, intraocular melanoma, pancreatic islet cell carcinoma (pancreatic pancreas) Endocrine gland), capage sarcoma, kidney cancer (renal cell cancer), pharyngeal cancer, leukemia, acute lymphoblastic leukemia, acute myeloid leukemia, chronic lymphocytic leukemia, chronic myeloid leukemia, hairy cell leukemia, lip and oral cancer , Fat sarcoma, liver cancer, non-small cell lung cancer, small cell lung cancer, lymphoma, AIDS-related lymphoma, Berkitt lymphoma, cutaneous T-cell lymphoma, Hodgkin lymphoma, non-Hodgkin lymphoma, primary central nervous system lymphoma, Waldenstraye Mumacroglobulinemia, malignant fibrous histiocytoma / osteosarcoma of bone, juvenile myelblastoma, melanoma, intraocular (eye) melanoma, Merkel cell carcinoma, adult malignant mesotheloma, juvenile mesentery Tumor, latent primary metastatic squamous cervical cancer, oral cancer, juvenile multiple endocrine tumor syndrome, multiple myeloma / plasma cell tumor, mycobacterial sarcoma, myelodystrophy syndrome, myeloid dysplasia / myeloid proliferative Disease, chronic myeloid leukemia, Adult acute myeloid leukemia, juvenile acute myeloid leukemia, multiple myeloma (cancer of the bone marrow), chronic myeloproliferative disease, nasal and sinus cancer, nasopharyngeal carcinoma, neuroblastoma, oral cancer, oropharyngeal cancer , Osteosarcoma / malignant fibrous histiocytoma of bone, ovarian cancer, epithelial ovarian cancer (surface epithelial stromal tumor), ovarian germ cell tumor, low-grade ovarian tumor, pancreatic cancer, pancreatic islet cell cancer, sinus and nasal cavity Cancer, parathyroid cancer, penis cancer, pharyngeal cancer, brown cell tumor, pine fruit stellate cell tumor, pine fruit embryo tumor, juvenile pine fruit blastoma and tent upper primitive nerve ectodermal tumor, pituitary gland Adenomas, plasma cell tumors / multiple myeloma, pleural lung blastoma, primary central nervous system lymphoma, prostate cancer, rectal cancer, renal cell carcinoma (kidney cancer), urinary tract cancer, retinal blastoma, juvenile lateral Crest myoma, salivary adenocarcinoma, Ewing tumor sarcoma, capage sarcoma, soft tissue sarcoma, uterine sarcoma, Cesarly syndrome, skin cancer (non-melanoma), skin cancer (melanoma), Merkel cell skin cancer, small bowel cancer, squamous epithelium Cancer, latent primary metastatic squamous cervical cancer, juvenile tent upper primitive neuroextradermal tumor, testis cancer, throat cancer, juvenile thoracic adenoma, thoracic adenoma and thoracic adenocarcinoma, thyroid cancer, juvenile thyroid cancer, urine Translocation epithelial cancer of the canal, gestational chorionic villus tumor, urinary tract cancer, endometrial uterine cancer, uterine sarcoma, vaginal cancer, juvenile tract and hypothalamic glioma, genital cancer, Waldenström macroglobulinemia, and Juvenile Wilms tumor (kidney cancer).

In a further preferred embodiment, the composition of the invention comprising the nucleic acid sequence of the invention as defined herein, or a plurality of the nucleic acid sequences of the invention as defined herein, for preparing a pharmaceutical composition or vaccine. , In particular for the purposes defined herein.

The pharmaceutical compositions or vaccines of the present invention may further be used for the treatment of a disease or disease, preferably a cancer or tumor disease as defined herein.

Also, according to the last aspect, the present invention provides a kit, especially parts of the kit. Such kits, particularly parts of the kit, typically, at least one nucleic acid sequence of the invention, a nucleic acid sequence of the invention, as defined herein, alone or in combination with additional components as defined herein. The pharmaceutical composition or vaccine of the present invention containing the above is included as a component. At least one nucleic acid sequence of the invention as defined herein is optionally combined with additional components as defined herein, said at least one nucleic acid of the invention comprising one or more other components. It is provided separately from at least one other part of the including kit (the first part of the kit). The pharmaceutical composition of the invention and / or the vaccine of the invention may be present, for example, in one or a different part of the kit. As an example, for example, at least one part of the kit comprises at least one nucleic acid sequence of the invention as defined herein and at least one additional part of the kit (at least one other configuration as defined herein). Elements) and may be included. For example, at least one other part of the kit may include at least one pharmaceutical composition or vaccine, or a portion thereof. For example, at least one part of the kit includes the nucleic acid sequence of the invention as defined herein, and at least one additional part of the kit (at least one other component as defined herein) of the kit. At least one additional part (at least one component of the pharmaceutical composition or vaccine of the invention, or pharmaceutical composition or vaccine of the invention as a whole) and at least one additional part of the kit (eg, at least 1). It may contain one pharmaceutical carrier or vehicle). If the kit or parts of the kit contain a plurality of nucleic acid sequences of the present invention, one component of the kit may contain only one, some, or all of the nucleic acid sequences of the present invention contained in the kit. In another embodiment, different / separate components of the kit may contain each / all nucleic acid sequences of the invention such that each component forms a part of the kit. Also, more than one nucleic acid may be included in the first component as a part of the kit, while one or more other (second, third) (providing one or more other parts of the kit). Etc.) The component may contain one or more nucleic acids of the invention, said nucleic acid being identical or partially identical to the first component. It may be different. The kit or parts of the kit are the administration of any of the nucleic acid sequences of the invention, the pharmaceutical compositions of the invention, or the vaccines of the invention, or components or parts thereof if the kit is prepared as parts of the kit. It may further include technical instructions with information on dosage.

In summary, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) A nucleic acid sequence containing or encoding a poly (A) sequence or a polyadenylation signal.
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, and more preferably CT-X antigen, non-X CT. Provided is a nucleic acid sequence that is a binding partner of an antigen, a tumor-specific antigen, or a fragment, variant, or derivative of the tumor antigen.

In a more preferred embodiment, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) A composition comprising at least one nucleic acid sequence comprising or encoding a poly (A) sequence or a polyadenylation signal.
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, and more preferably CT-X antigen, non-X CT. It relates to a composition which is a binding partner of an antigen, a tumor-specific antigen, or a fragment, variant, or derivative of the tumor antigen, wherein the nucleic acid sequence encodes a different peptide or protein, respectively.

The composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant as defined herein. The composition may be used as a vaccine or for treating a disease associated with cancer or tumor.

In a more preferred embodiment, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) At least two, preferably two or more, more preferably multiple (typically more than one, more than two, more than three) containing or encoding a poly (A) sequence or polyadenylation signal. A composition comprising a nucleic acid sequence (meaning more than, more than 4, more than 5, more than 5, or more than 10 nucleic acids, eg, 2-10, preferably 2-5) nucleic acids. There,
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, and more preferably CT-X antigen, non-X CT. Provided is a composition which is a binding partner of an antigen, a tumor-specific antigen, or a fragment, variant, or derivative of the tumor antigen, wherein the nucleic acid sequence encodes a different peptide or protein, respectively.

The composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant as defined herein. The composition may be used as a vaccine or for treating a disease associated with cancer or tumor.

In a more preferred embodiment, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) At least two, preferably two or more, more preferably multiple (typically more than one, more than two, more than three) containing or encoding a poly (A) sequence or polyadenylation signal. A composition comprising a nucleic acid sequence (meaning more than, more than 4, more than 5, more than 5, or more than 10 nucleic acids, eg, 2-10, preferably 2-5) nucleic acids. There,
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, and more preferably CT-X antigen, non-X CT. An antigen-binding partner, a tumor-specific antigen, or a fragment, variant, or derivative of the tumor antigen, wherein the nucleic acid sequences encode different peptides or proteins, preferably various types of the nucleic acid sequences. Compositions that encode different peptides or proteins, preferably different tumor antigens, are provided.

The composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant as defined herein. The composition may be used as a vaccine or for treating a disease associated with cancer or tumor.

In a more preferred embodiment, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) At least two, preferably two or more, more preferably multiple (typically more than one, more than two, more than three) containing or encoding a poly (A) sequence or polyadenylation signal. A composition comprising a nucleic acid sequence (meaning more than, more than 4, more than 5, more than 5, or more than 10 nucleic acids, eg, 2-10, preferably 2-5) nucleic acids. There,
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, more preferably CT-X antigen, non-X CT. A binding partner of an antigen, a tumor-specific antigen, or a fragment, variant, or derivative of said tumor antigen, each of which encodes a different peptide or protein, preferably a variety of said nucleic acid sequences. Different peptides or proteins, preferably different tumor antigens, and more preferably one of the nucleic acid sequences contained are PSA, PSMA, PSCA, STEAP-1, NY-ESO-1, 5T4, A composition encoding Survival, MAGE-C1, or MAGE-C2 is provided.

The composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant as defined herein. The composition may be used as a vaccine or for treating a disease associated with cancer or tumor.

In some embodiments, it may be preferable that the nucleic acid sequence does not encode NY-ESO1 only if the composition contains only one nucleic acid sequence.

In a more preferred embodiment, the present invention
a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) A composition comprising at least two nucleic acid sequences comprising or encoding a poly (A) sequence or a polyadenylation signal.
The peptide or protein comprises a tumor antigen, a fragment, variant, or derivative of the tumor antigen, preferably the tumor antigen is a melanin-forming cell-specific antigen, a cancer-testis antigen, or a tumor-specific antigen. , Preferred are CT-X antigen, non-X CT antigen, CT-X antigen binding partner, non-X CT antigen binding partner, or tumor-specific antigen, and more preferably CT-X antigen, non-X CT. An antigen binding partner, a tumor-specific antigen, or a fragment, variant, or derivative of the tumor antigen, wherein the nucleic acid sequences encode different peptides or proteins, and at least one of the nucleic acid sequences. 5T4, 707-AP, 9D7, AFP, AlbZIP HPG1, alpha-5-beta-1-integrin, alpha-5-beta-6-integrin, alpha-actinine-4 / m, alpha-methylacyl coenzyme A racemase, ART -4, ARTC1 / m, B7H4, BAGE-1, BCL-2, bcr / abl, beta-catenin / m, BING-4, BRCA1 / m, BRCA2 / m, CA15-3 / CA27-29, CA19-9 , CA72-4, CA125, carreticulin, CAMEL, CASP-8 / m, antigen B, catheter L, CD19, CD20, CD22, CD25, CDE30, CD33, CD4, CD52, CD55, CD56, CD80, CDC27 / m, CDK4 / m, CDKN2A / m, CEA, CLCA2, CML28, CML66, COA-1 / m, antigen-like protein, collagen XXIII type, COX-2, CT-9 / BRD6, Cten, cyclone B1, cyclone D1, cyp- B, CYPB1, DAM-10, DAM-6, DEK-CAN, EFTUD2 / m, EGFR, ELF2 / m, EMMPRIN, EpCam, EphA2, EphA3, ErbB3, ETV6-AML1, EZH2, FGF-5, FN, Frau- 1, G250, GAGE-1, GAGE-2, GAGE-3, GAGE-4, GAGE-5, GAGE-6, GAGE7b, GAGE-8, GDEP, GnT-V, gp100, GPC3, GPNMB / m, HAGE, HAST-2, Hepsin, Her2 / neu, HERV-K-MEL, HLA-A ^* 0201-R17I, HLA-A11 / m, HLA-A2 / m, HNE, Homeobox NKX3.1, HO M-TES-14 / SCP-1, HOM-TES-85, HPV-E6, HPV-E7, HSP70-2M, HST-2, hTERT, iCE, IGF-1R, IL-13Ra2, IL-2R, IL- 5, immature laminin receptor, calicrane 2, calicrane 4, kinase 0205, kinase 0205 / m, KK-LC-1, K-Ras / m, LAGE-A1, LDLR-FUT, MAGE-A1, MAGE-A2, MAGE-A3, MAGE-A4, MAGE-A6, MAGE-A9, MAGE-A10, MAGE-A12, MAGE-B1, MAGE-B2, MAGE-B3, MAGE-B4, MAGE-B5, MAGE-B6, MAGE- B10, MAGE-B16, MAGE-B17, MAGE-C1, MAGE-C2, MAGE-C3, MAGE-D1, MAGE-D2, MAGE-D4, MAGE-E1, MAGE-E2, MAGE-F1, MAGE-H1, MAGEL2, Mammaglobin A, MART-1 / melan-A, MART-2, MART-2 / m, Substrate protein 22, MC1R, M-CSF, ME1 / m, Osteopontin, MG50 / PXDN, MMP11, MN / CA IX Antigens, MRP-3, MUC-1, MUC-2, MUM-1 / m, MUM-2 / m, MUM-3 / m, myosin class I / m, NA88-A, N-acetylglucosaminyl kinase- V, Neo-PAP, Neo-PAP / m, NFYC / m, NGEP, NMP22, NPM / ALK, N-Ras / m, NSE, NY-ESO-B, OA1, OFA-iLRP, OGT, OGT / m, OS-9, OS-9 / m, osteocalcin, osteopontin, p15, p190 minor bcr-abl, p53, p53 / m, PAGE-4, PAI-1, PAI-2, PAP, PART-1, PATE, PDEF, Pim-1 Kinase, Pin-1, Pml / PAR Alpha, POTE, PRAME, PRDX5 / m, Prostain, Proteinase 3, PSA, PSCA, PSGR, PSM, PSMA, PTPRK / m, RAGE-1, RBAF600 / m, RHAMM / CD168, RU1, RU2, S-100, SAGE, SART-1, SART-2, SART-3, SCC, SIRT2 / m, Sp17, SSX-1, SSX-2 / HOM-MEL-40, SSX- 4, STAMP-1, STEA P-1, Survival, Survival-2B, SYT-SSX-1, SYT-SSX-2, TA-90, TAG-72, TARP, TEL-AML1, TGF Beta, TGF Beta RII, TGM-4, TPI / m , TRAG-3, TRG, TRP-1, TRP-2 / 6b, TRP / INT2, TRP-p8, tyrosinase, UPA, VEGFR1, VEGFR-2 / FLK-1, WT1, and immunoglobulin iditypes of lymphocytic cells or T cell receptor iditypes of lymphoid cells, or fragments, variants, or derivatives of the tumor antigen, preferably survivin or homologs thereof, antigens from the MAGE family or binding partners thereof, or fragments, variants of the tumor antigen. , Or a composition encoding a derivative.

The composition may further comprise a pharmaceutically acceptable carrier and / or a pharmaceutically acceptable adjuvant as defined herein. The composition may be used as a vaccine or for treating a disease associated with cancer or tumor.

In the present invention, alternatives and embodiments of different characteristics may be combined with each other unless otherwise indicated. Furthermore, the term "including" shall not be construed as meaning "consisting of" unless otherwise specified. However, in the present invention, the term "including" may be replaced with the term "consisting of", where applicable.

The following examples are intended to further illustrate the invention and are not to be construed as limiting the invention to it.

1. 1. Preparation of histone stem-loop consensus sequence Prior to the experiment, the histone stem-loop consensus sequence was determined based on the histone stem-loop consensus sequence of metazoans and protozoans. Lopez et al. (Davilla Lopez, M., & Samuelsson, T. (2008), RNA (New York, NY), 14) identified a large number of native histone stem-loop sequences by searching for genomic and expression sequence tags. Sequences were taken from the addendum provided by (1), 1-10. Doi: 10.261 / rna.782308). First, the entire sequence derived from metazoans and protozoans (4,001 sequences), the entire sequence derived from protozoa (131 sequences), the entire sequence derived from metazoans (3,870 sequences), or the entire sequence derived from vertebrates. (1,333 sequences) or all human-derived sequences (84 sequences) were grouped and aligned. The number of nucleotides present was then determined for all positions. Based on the table thus obtained, consensus sequences for five different groups of sequences representing the nucleotides present in the entire analyzed sequence were created. In addition, a more limited consensus sequence was obtained to phase out the conserved nucleotides.

2. Preparation of DNA template Following the T7 promoter, the GC-rich sequence encoding the Kita-American firefly luciferase (ppLuc (GC)), the central portion (ag) of the 3'untranslated region (UTR) of alphaglobin, and poly ( A) A vector for in vitro transcription containing the sequence was constructed. To obtain mRNA terminating with the A64 poly (A) sequence, a restriction enzyme site used to linearize the vector prior to in vitro transcription was placed immediately after the poly (A) sequence. Therefore, the mRNA obtained from this vector by in vitro transcription was named "ppLuc (GC) -ag-A64".

By linearizing this vector at another restriction enzyme site prior to in vitro transcription, mRNA with A64 3'extended by additional nucleotides or A64-deficient mRNA could be obtained. In addition, the original vector was modified to include another sequence. In summary, in vitro transcription gave the following mRNAs from these vectors (mRNA sequences are shown in Figures 6-17).
ppLuc (GC) -ag (SEQ ID NO: 43)
ppLuc (GC) -ag-A64 (SEQ ID NO: 44)
ppLuc (GC) -ag-histone SL (SEQ ID NO: 45)
ppLuc (GC) -ag-A64-histone SL (SEQ ID NO: 46)
ppLuc (GC) -ag-A120 (SEQ ID NO: 47)
ppLuc (GC) -ag-A64-ag (SEQ ID NO: 48)
ppLuc (GC) -ag-A64-aCPSL (SEQ ID NO: 49)
ppLuc (GC) -ag-A64-Polio CL (SEQ ID NO: 50)
ppLuc (GC) -ag-A64-G30 (SEQ ID NO: 51)
ppLuc (GC) -ag-A64-U30 (SEQ ID NO: 52)
ppLuc (GC) -ag-A64-SL (SEQ ID NO: 53)
ppLuc (GC) -ag-A64-N32 (SEQ ID NO: 54)

Furthermore, DNA plasmid sequences encoding the tumor antigens NY-ESO-1, survivin, and MAGE-C1 were prepared as described above.
In summary, in vitro transcription gave the following mRNAs from these vectors (mRNA sequences are shown in Figures 18-21).
NY-ESO-1 (GC) -ag-A62-C30 (SEQ ID NO: 55)
NY-ESO-1 (GC) -ag-A62-C30-Histone SL
(SEQ ID NO: 56)
Survivin (GC) -ag-A62-C30-histone SL (SEQ ID NO: 57)
MAGE-C1 (GC) -ag-A64-C30-Histone SL
(SEQ ID NO: 58)

3. 3. In vitro transcription The DNA template according to Example 2 was linearized and transcribed in vitro using T7-polymerase. The DNA template was then degraded by DNase treatment. Addition of excess N7-methyl-guanosine-5'-triphosphate-5'-guanosine to the transcription reaction gave the total mRNA transcript containing the 5'-CAP structure. The mRNA thus obtained was purified and resuspended in water.

4. Enzymatic adenylation of mRNA Two mRNAs were enzymatically adenylated:
ppLuc (GC) -ag-A64 and ppLuc (GC) -ag-histone SL.
To this end, RNA was incubated with E. coli poly (A) -polymerase and ATP (Poly (A) polymerase tailing kit, Epicenter, Madison, USA) according to the manufacturer's instructions. The mRNA containing the elongated poly (A) sequence was purified and resuspended in water. The length of the poly (A) sequence was determined via agarose gel electrophoresis. The starting mRNA was elongated by about 250 adenilates and the resulting mRNAs were named ppLuc (GC) -ag-A300 and ppLuc (GC) -ag-histone SL-A250, respectively.

5. Luciferase expression by mRNA electroporation HeLa cells were trypsinized and washed with opti-MEM. 0.5 μg of mRNA encoding ppLuc was electroporated into ^{1 × 10 5} cells in 200 μL of opti-MEM. As a control, mRNA not encoding ppLuc was separately electroporated. Electroporated cells were seeded in 1 mL of RPMI 1640 medium in a 24-well plate. After 6 hours, 24 hours, or 48 hours of transfection, the medium is aspirated and lysis buffer 200 μL (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM). Cells were lysed in DTT (1 mM PMSF). The lysate was stored at −20 ° C. until ppLuc activity was measured.

6. Luciferase expression by mRNA lipofection HeLa cells were seeded on 96-well plates to a density of ^{2 × 10 4 cells per well.} The next day, cells were washed with opti-MEM and then transfected with 0.25 μg of lipofectin complexed ppLuc coding mRNA in 150 μL of opti-MEM. As a control, mRNA not encoding ppLuc was separately lipofected. In some wells, 6 hours after the start of transfection, opti-MEM was aspirated to lyse cells in 200 μL of lysis buffer. In the remaining wells, opti-MEM was then replaced with PTMI1640 medium. In these wells, 24 hours or 48 hours after the start of transfection, medium was aspirated and cells were lysed in 200 μL of lysis buffer. The lysate was stored at −20 ° C. until ppLuc activity was measured.

7. Luciferase measurement Using 50 μL of lysate and 200 μL of luciferin buffer (25 mM glycylglycine, pH 7.8 (NaOH), 15 mM Then ₄ , 2 mM ATP, 75 μM luciferin), a relative light unit by a BioTek Synergy HT plate reader with a measurement time of 5 seconds. The ppLuc activity was measured as (RLU). The ratio RLU was calculated by subtracting the control RNA RLU from the total RLU.

8. Luciferase expression by transdermal mRNA injection (luciferase expression in vivo)
Mice were anesthetized with a mixture of Ronpun and Ketabet. Each mRNA encoding ppLuc was transdermally injected (0.5 μg of mRNA in 50 μL per injection). As a control, mRNA not encoding ppLuc was injected separately. 16 hours after injection, mice were sacrificed and tissue was harvested. Tissue samples were snap frozen in liquid nitrogen and dissolved in tissue solution (25 mM Tris, pH 7.5 (HCl), 2 mM EDTA, 10% glycerol, 1% Triton X-100, 2 mM DTT, 1 mM PMSF). It was dissolved in Qiagen). The sample was then centrifuged at 4 ° C. for 10 minutes at 13,500 rpm. The lysate was stored at −80 ° C. until ppLuc activity was measured (see 7. Luciferase Expression).

9. NY-ESO-1 expression HeLa cells by electroporation of mRNA were trypsinized and washed with opti-MEM. 10 μg of NY-ESO-1 encoding mRNA was electroporated into ^{2 × 10 5} cells in 200 μL of opti-MEM. Cells obtained from three electroporations were combined and seeded in 2 mL of RPMI 1640 medium in a 6-well plate. Twenty-four hours after transfection, cells were collected and transferred to a 96-well V-bottom plate (2 wells per mRNA). The cells were washed with phosphate buffered saline (PBS) and permeabilized with 200 μL Cytofix / Cytoperm (Becton Dickinson (BD)) per well. After 15 minutes, the cells were washed with PermWash (BD). The cells were then incubated with mouse anti-NY-ESO-1 IgG1 or isotype control (20 μg / mL) for 1 hour at room temperature. The cells were washed twice again with PermWash. The cells were then incubated with a 1: 500 dilution of Alexa-647 bound goat anti-mouse IgG at 4 ° C. for 1 hour. Finally, the cells were washed twice with PermWash. The cells were resuspended in 200 μL of buffer (PBS, 2% FCS, 2 mM EDTA, 0.01% sodium azide). NY-ESO-1 expression was quantified by flow cytometry as central fluorescence intensity (MFI).

10. Induction of Anti-NY-ESO-1 Antibodies by Vaccination with mRNA C57BL / 6 mice were transdermally vaccinated with mRNA encoding NY-ESO-1 complexed with protamine (within 14 days). 5 times). The control mouse was buffered. Eight days after the last vaccination by ELISA, the levels of NY-ESO-1-specific antibodies in vaccinated and control mice were analyzed. A 96-well ELISA plate (Nunc) was coated with 100 μL of recombinant NY-ESO-1 protein 10 μg / mL per well for 16 hours at 4 ° C. The plate was washed twice with wash buffer (PBS, 0.05% Tween-20). The plate was then incubated with a blocking buffer (PBS, 0.05% Tween-20, 1% BSA) at 37 ° C. for 2 hours to block non-specific binding. After blocking, 100 μL of serially diluted mouse serum per well was added and incubated at room temperature for 4 hours. The plate was then washed 3 times with wash buffer. Next, biotinylated rat anti-mouse IgG2a [b] detection antibody (BD Biosciences) (100 μL per well) diluted 1: 600 in blocking buffer was bound at room temperature for 1 hour. The plates were washed again in wash buffer three times and then incubated with 100 μL of horseradish peroxidase-conjugated streptavidin per well for 30 minutes at room temperature. After washing 4 times in the wash buffer, 100 μL of 3,3', 5,5'-tetramethylbenzidine (Thermo Scientific) was added per well. When the color changed, 100 μL of 20% sulfuric acid was added per well. Absorbance was measured at 405 nm.

11. Induction of anti-survival antibody by mRNA vaccination C57BL / 6 mice were transdermally vaccinated with mRNA encoding survivin complexed with protamine (5 times within 14 days). The control mouse was buffered. Eight days after the last vaccination by ELISA, the levels of survivin-specific antibodies in vaccinated and control mice were analyzed. A 96-well ELISA plate (Nunc) was coated with 10 μg / mL of 100 μL of recombinant survivin protein per well for 16 hours at 4 ° C. The plate was washed twice with wash buffer (PBS, 0.05% Tween-20). The plate was then incubated with a blocking buffer (PBS, 0.05% Tween-20, 1% BSA) at 37 ° C. for 2 hours to block non-specific binding. After blocking, 100 μL of serially diluted mouse serum per well was added and incubated at room temperature for 4 hours. The plate was then washed 3 times with wash buffer. Next, biotinylated rat anti-mouse IgG2a [b] detection antibody (BD Biosciences) (100 μL per well) diluted 1: 600 in blocking buffer was bound at room temperature for 1 hour. The plates were washed again in wash buffer three times and then incubated with 100 μL of horseradish peroxidase-conjugated streptavidin per well for 30 minutes at room temperature. After washing 4 times in the wash buffer, 100 μL of 3,3', 5,5'-tetramethylbenzidine (Thermo Scientific) was added per well. When the color changed, 100 μL of 20% sulfuric acid was added per well. Absorbance was measured at 405 nm.

12. Induction of Anti-MAGE-C1 Antibodies by Vaccination with mRNA C57BL / 6 mice were transdermally vaccinated with mRNA encoding MAGE-C1 complexed with protamine (5 times within 14 days). The control mouse was buffered. Eight days after the last vaccination by ELISA, the levels of MAGE-C1-specific antibody in vaccinated and control mice were analyzed. A 96-well ELISA plate (Nunc) was coated at 4 ° C. for 16 hours with 100 μL of recombinant MAGE-C1 protein per well at 10 μg / mL. The plate was washed twice with wash buffer (PBS, 0.05% Tween-20). The plate was then incubated with a blocking buffer (PBS, 0.05% Tween-20, 1% BSA) at 37 ° C. for 2 hours to block non-specific binding. After blocking, 100 μL of serially diluted mouse serum per well was added and incubated at room temperature for 4 hours. The plate was then washed 3 times with wash buffer. Next, biotinylated rat anti-mouse IgG2a [b] detection antibody (BD Biosciences) (100 μL per well) diluted 1: 600 in blocking buffer was bound at room temperature for 1 hour. The plates were washed again in wash buffer three times and then incubated with 100 μL of horseradish peroxidase-conjugated streptavidin per well for 30 minutes at room temperature. After washing 4 times in the wash buffer, 100 μL of 3,3', 5,5'-tetramethylbenzidine (Thermo Scientific) was added per well. When the color changed, 100 μL of 20% sulfuric acid was added per well. Absorbance was measured at 405 nm.

13. Detection of Antigen-Specific Cell Immune Response (T Cell Immune Response) by ELISPOT C57BL / 6 mice were transdermally vaccinated with mRNA encoding MAGE-C1 complexed with protamine (within 14 days). 5 times). The control mouse was buffered. Mice were sacrificed one week after the last vaccination, the spleen was removed and spleen cells were isolated. The spleen cells are restimulated for 7 days in the presence of the antigen-derived peptide (peptide library) or co-incubated with dendritic cells generated from the bone marrow cells of native syngeneic mice to encode the antigen. The mRNA was electroporated. INF gamma secretion was measured after restimulation to determine the antigen-specific cellular immune response. To detect INF gamma, a coating buffer (0.1 M carbonate-bicarbonate buffer, pH 9.6, 10.59 g / L Na2CO3, 8.4 g / L) containing an antibody against INFγ (BD Harmingen, Heidelberg, Germany). Incubate coated multiscreen plates (Millipore) with NaHCO3) overnight. The stimulant and effector cells are incubated together in a plate at a ratio of 1:20 for 24 hours. The plate is washed with 1 x PBS and incubated with biotin-conjugated secondary antibody. After washing with 1 × PBS / 0.05% Tween-20, the substrate (5-bromo-4-chloro-3-indrill phosphate / nitroblue tetrazolium liquid substrate system (Sigma Aldrich, Taufkirchen, Germany)) was subjected to the above. When added to the plate, substrate conversion could be visually detected.

14. Results 14.1 Histone stem-loop sequence:
To characterize the histon stem loop sequence, a sequence derived from metazoans and protozoans (4,001 sequences), a sequence derived from protozoa (131 sequences), or a sequence derived from metazoans (3,870 sequences). , Or vertebrate-derived sequences (1,333 sequences), or human-derived sequences (84 sequences) were grouped and aligned. The number of nucleotides present was then determined for all positions. Based on the table thus obtained, consensus sequences for five different groups of sequences representing the nucleotides present in the entire analyzed sequence were created. The consensus sequence of metazoans and protozoa conserved three nucleotides, T / U in the loop and G and C in the stem, which form base pairs. Structurally, a 6-base pair stem and a 4-nucleotide loop are typically formed. However, different structures are also common: of the 84 human histone stemloops, 2 contain only 5 nucleotide stems containing 4 base pairs and 1 mismatch. Another human histone stem-loop contains a stem with only 5 base pairs. Four additional human histone stem-loops contain a 6 nucleotide long stem, each containing one mismatch at three different positions. In addition, each of the four human histone stemloops contains one wobble base pair at two different positions. It is considered that the loop does not have to be exactly 4 nucleotides in length as in the 5-nucleotide loop identified by D. discoidium.

In addition to the consensus sequences representing the nucleotides present in the entire sequence analyzed, a more limited consensus sequence was also obtained to phase out the conserved nucleotides. In summary, the following sequences were obtained:
(Cons): Represents all nucleotides present (99%): Represents at least 99% of all nucleotides present (95%): Represents at least 95% of all nucleotides present (90%) ): Represents at least 90% of all nucleotides present

The results of histone stem-loop sequence analysis are summarized in Tables 1-5 below (see also Figures 1-5):

The abbreviations used here are defined as follows:

14.2 The combination of poly (A) and histone SL synergistically increases protein expression from mRNA To investigate the effect of the combination of poly (A) and histone SL on protein expression from mRNA, alphaglobin 3 We synthesized mRNAs containing various sequences in'-UTR 3': mRNAs that are terminated at exactly 3'-UTR 3'and lack both poly (A) sequences and histone SLs, or A64 poly (A). An mRNA containing either the sequence or histone SL, or an mRNA containing both the A64 poly (A) sequence and histone SL in 3'of the 3'-UTR. The mRNA encoding luciferase or control mRNA was electroporated into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after transfection (see Table 6 and FIG. 22 below).

Almost no luciferase was expressed from mRNA having neither poly (A) sequence nor histone SL. Both the poly (A) sequence or histone SL increased luciferase levels to a similar extent. These mRNAs yielded much higher luciferase levels than mRNAs lacking both poly (A) and histone SL. However, surprisingly, the combination of poly (A) and histone SL increased luciferase levels even more strongly than those found in any of the individual elements. The magnitude of the increase in luciferase levels by combining poly (A) and histone SL in the same mRNA demonstrates that they act synergistically.

The signal obtained from poly (A) -histone SL mRNA (+ / +) is the signal obtained from histone SL mRNA (-/ +) and the signal obtained from poly (A) mRNA (+/-). The synergistic effect of poly (A) and histone SL was quantified by dividing by the sum (see Table 7 below).

The coefficients thus calculated are obtained from the mRNA combining poly (A) and histone SL than would be expected if the effects of poly (A) and histone SL were purely additive. Shows how high the luciferase level is. Luciferase levels obtained from mRNA in combination with poly (A) and histone SL were up to 16.6 times higher when these effects were purely additive. This result confirms that the combination of poly (A) and histone SL affects a significant synergistic increase in protein expression.

14.3 The combination of poly (A) and histone SL increases protein expression from mRNA regardless of its order. The effect of the combination of poly (A) and histone SL is the length of the poly (A) sequence. And it may depend on the order of poly (A) and histone SL. Therefore, mRNA in which the poly (A) sequence was extended and mRNA in which the order of poly (A) and histone SL was reversed were synthesized. The two mRNAs contained the A120 poly (A) sequence or the A300 poly (A) sequence in the 3'of the 3'-UTR. One additional mRNA contained the histone SL first in the 3'-UTR 3', followed by the A250 poly (A) sequence. The mRNA encoding luciferase or the control mRNA was lipofected into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after the start of transfection (see Table 8 and FIG. 23 below).

Equivalent luciferase levels were obtained from the A64 poly (A) sequence and histone SL. Consistent with previous experiments, the combination of A64 and histone SL strongly increased luciferase levels many times higher than those found in any of the individual elements. The magnitude of the increase in luciferase levels by combining poly (A) and histone SL in the same mRNA demonstrates that they act synergistically. As mentioned above, the synergistic effect of A64 and histone SL was quantified based on the luciferase levels of A64-histone SL mRNA, A64 mRNA, and histone SL mRNA (see Table 9 below). Luciferase levels obtained from mRNA in combination with A64 and histone SL were up to 61.7 times higher when the effects of poly (A) and histone SL were purely additive.

In contrast, extending the length of the poly (A) sequence from A64 to A120, or A300 resulted in only a slight increase in luciferase levels (see Table 8 and FIG. 19). Also, mRNA with the longest poly (A) sequence (A300) was compared to mRNA (histone SL-A250) in which a poly (A) sequence of similar length was combined with histone SL. Compared to A64-histone SL mRNA, this mRNA has a longer poly (A) sequence and the order of histone SL and poly (A) is reversed. The combination of A250 and histone SL strongly increased luciferase levels many times higher than those found with histone SL or A300. In addition, as described above, the synergistic effect between A250 and histone SL was quantified by comparing the RLU obtained from histone SL-A250 mRNA with the RLU obtained from RLU + histone SL mRNA obtained from A300 mRNA (Table). 10). Luciferase levels obtained from mRNA in combination with A250 and histone SL were up to 17.0 times higher when the effects of poly (A) and histone SL were purely additive.

In summary, for substantially different lengths of poly (A), the combination of histone SL and poly (A) is high for protein expression from mRNA, regardless of the order of poly (A) and histone SL. It has been proven to show a synergistic effect.

14.4 Increased protein expression by combination of poly (A) and histone SL is specific Investigate whether the effect of the combination of poly (A) and histone SL on protein expression from mRNA is specific To be used, mRNAs containing a combination of poly (A) and another sequence were synthesized: each of these mRNAs contained one of seven different sequences in 3'of A64. The mRNA encoding luciferase or control mRNA was electroporated into HeLa cells. Luciferase levels were measured 6 hours, 24 hours, and 48 hours after transfection (see Table 11 and FIG. 24 below).

Equivalent luciferase levels were obtained from the poly (A) sequence and histone SL. Also, the combination of poly (A) and histone SL strongly increased luciferase levels many times higher than those found in any of the individual elements. Therefore, they acted synergistically. In contrast, the combination of poly (A) with any of the other sequences had no effect on luciferase levels compared to mRNA containing only the poly (A) sequence. Therefore, the combination of poly (A) and histone SL synergistically increases protein expression from mRNA, a specific effect.

14.5 Poly (A) plus histone SL synergistically increases protein expression from mRNA in vivo Examining the effect of the combination of poly (A) and histone SL on protein expression from mRNA in vivo To be used, mice were transdermally injected with mRNA or control mRNA encoding a luciferase containing various sequences in alphaglobin 3'-UTR 3': the mRNA was A64 poly in 3'-UTR 3'. It contained both the (A) sequence, histone SL, or the A64 poly (A) sequence and histone SL. Luciferase levels were measured 16 hours after injection (see Table 12 and Figure 25 below).

Luciferase was expressed from mRNA with histone SL or poly (A) sequence. However, the combination of poly (A) and histone SL increased luciferase levels even more strongly than those found in any of the individual elements. The magnitude of the increase in luciferase levels by combining poly (A) and histone SL in the same mRNA demonstrates that they act synergistically.

The signal obtained from poly (A) -histone SL mRNA (+ / +) is the signal obtained from histone SL mRNA (-/ +) and the signal obtained from poly (A) mRNA (+/-). The synergistic effect of poly (A) and histone SL was quantified by dividing by the sum (see Table 13 below).

The coefficients thus calculated are obtained from the mRNA combining poly (A) and histone SL than would be expected if the effects of poly (A) and histone SL were purely additive. Shows how high the luciferase level is. Luciferase levels obtained from mRNA in combination with poly (A) and histone SL were eight-fold higher than when these effects were purely additive. This result confirms that the combination of poly (A) and histone SL affects a significant synergistic increase in protein expression in vivo.

14.6 The combination of poly (A) and histone SL increases the expression of NY-ESO-1 protein from mRNA To investigate the effect of the combination of poly (A) and histone SL on protein expression from mRNA. , An mRNA encoding NY-ESO-1 having various sequences in alphaglobin 3'-UTR 3'was synthesized. The mRNA contained the A64 poly (A) sequence or both the A64 poly (A) and histone SL in the 3'of the 3'-UTR. The mRNA encoding NY-ESO-1 was electroporated into HeLa cells. NY-ESO-1 levels were measured 24 hours after transfection by flow cytometry (see Table 14 and FIG. 26 below).

NY-ESO-1 was expressed from mRNA having only the poly (A) sequence. However, surprisingly, the combination of poly (A) and histone SL strongly increased NY-ESO-1 levels many times higher than those found in the poly (A) sequence alone.

14.7 The combination of poly (A) and histon SL increases the level of antibody induced by mRNA vaccination. Poly (A) and histon SL to the induction of antibody induced by mRNA vaccination. To investigate the effect of the combination, an mRNA encoding NY-ESO-1, which has various sequences in alphaglobin 3'-UTR 3', which is complexed with protamine, was transdermally administered to C57BL / 6 mice. Vaccinated. The mRNA contained the A64 poly (A) sequence or both the A64 poly (A) and histone SL in the 3'of the 3'-UTR. Levels of NY-ESO-1 specific antibody in vaccinated and control mice were analyzed by ELISA using serial dilutions of serum (see Table 15 and FIG. 27 below).

Anti-NY-ESO-1 IgG2a [b] was induced by mRNA having only the poly (A) sequence. However, surprisingly, the combination of poly (A) and histone SL strongly increased anti-NY-ESO-1 IgG2a [b] levels many times higher than those found in the poly (A) sequence alone. ..

Claims (25)

a) A coding region encoding at least one peptide or protein, and
b) With at least one histone stemloop,
c) A nucleic acid that contains or encodes a poly (A) sequence or a polyadenylation signal.
The peptide or protein comprises the tumor antigen K-Ras / m or a fragment, variant, or derivative thereof.
The fragment, variant, or derivative is a nucleic acid characterized by having at least 6 amino acids and having the same antigenic properties as full-length native K-Ras / m. The nucleic acid of claim 1, wherein at least one histone stem-loop is heterologous to the coding region encoding at least one peptide or protein. The nucleic acid according to claim 1 or 2, wherein the coding region does not encode a reporter protein or a marker or a selected protein. The nucleic acid according to any one of claims 1 to 3, wherein the nucleic acid is RNA. The nucleic acid according to any one of claims 1 to 4, wherein the nucleic acid is mRNA. The nucleic acid according to any one of claims 1 to 5, wherein at least one histone stem loop is selected from the following formula (I) or (II):
Equation (I) (Stem-loop array without stem boundary elements):

Formula (II) (stem loop array including stem boundary elements):

(During the ceremony,
Stem 1 boundary element or stem 2 boundary element N _1-6 is a continuous array of 1 to 6 N, each N being independently selected from A, U, T, G, and C. Nucleotides, or selected from these nucleotide analogs,
Stem 1 [N _0-2 GN _3-5 ] is a contiguous sequence of 5 to 7 nucleotides that is inversely complementary or partially inversely complementary to element stem 2. N _0-2 is a contiguous sequence of 0 to 2 N, each N independently of each other, from nucleotides selected from A, U, T, G, and C, or from these nucleotide analogs. Selected, N _3-5 is a contiguous sequence of 3-5 N, each N being a nucleotide selected from A, U, T, G, and C independently of each other, or any of these. Selected from nucleotide analogs, G is guanosine or an analog thereof and may optionally be substituted with cytidine or an analog thereof, provided that cytidine, its complementary nucleotide in stem 2, is also substituted with guanosine. Being done
The loop sequence [N _0-4 (U / T) N _0-4 ] is a continuous sequence of 3 to 5 nucleotides located between the element stem 1 and the stem 2, and each N _{0- 4} is a contiguous sequence of 0-4 N independently of each other, and each N is a nucleotide selected from A, U, T, G, and C independently of each other, or nucleotides thereof. Selected from analog, U / T represents uridine or optionally thymidine,
Stem 2 [N _3-5 CN _0-2 ] is a contiguous sequence of 5 to 7 nucleotides that is inversely complementary or partially inversely complementary to element stem 1. N _3-5 is a contiguous sequence of 3 to 5 N, each N independently of each other, from nucleotides selected from A, U, T, G, and C, or from these nucleotide analogs. Selected, N _0-2 is a contiguous sequence of 0 to 2 N, each N being a nucleotide selected from A, U, T, G, and C independently of each other, or any of these. Selected from nucleotide analogs, C is cytidine or an analog thereof and may optionally be substituted with guanosine or an analog thereof, provided that guanosine, its complementary nucleotide in stem 1, is also substituted with cytidine. Being done
Stem 1 and stem 2 can be base paired with each other to form an inverse complementary sequence, and the base pairing can occur between stem 1 and stem 2, or the stem 1 and stem 2 are base paired with each other. Incomplete base pairing can occur between stem 1 and stem 2). The nucleic acid according to claim 6, wherein at least one histone stem-loop is selected from at least one of the following formulas (Ia) or (IIa):
Equation (Ia) (Stemloop array without stem boundary elements):

Equation (IIa) (stem loop array including stem boundary elements):

.. The nucleic acid according to any one of claims 1 to 7, wherein the poly (A) sequence comprises a sequence of 25 to 400 adenosine nucleotides. The nucleic acid according to any one of claims 1 to 8, wherein the polyadenylation signal comprises a consensus sequence: NN (U / T) ANA. The nucleic acid according to any one of claims 1 to 9, wherein the polyadenylation signal comprises a consensus sequence: AAA (U / T) AAA or A (U / T) (U / T) AAA. The nucleic acid according to any one of claims 1 to 10, wherein the nucleic acid is a modified nucleic acid. The nucleic acid according to any one of claims 1 to 11, wherein the nucleic acid is a stabilized nucleic acid. The nucleic acid according to claim 11 or 12, wherein the G / C content of the coding region encoding at least one peptide or protein of the modified nucleic acid is increased as compared with the G / C content of the coding region of the wild-type nucleic acid. .. The nucleic acid according to claim 13, wherein the amino acid sequence encoded by the modified nucleic acid is not modified as compared with the amino acid sequence encoded by the wild-type nucleic acid. A composition comprising at least one of the nucleic acids according to any one of claims 1 to 14. The composition of claim 15, wherein the composition comprises at least two nucleic acids, each of which encodes a different peptide or protein. Each of the nucleic acids encodes a different tumor antigen or fragment, variant, or derivative thereof.
The composition according to claim 16, wherein the fragment, variant, or derivative has at least 6 amino acids and has the same antigenic properties as a full-length native tumor antigen. From claim 15, one of the nucleic acids contained encodes PSA, PSMA, PSCA, STEAP-1, NY-ESO-1, 5T4, Survivin, MAGE-C1, or MAGE-C2. The composition according to any one of 17. A kit or part of a kit comprising at least one nucleic acid according to any one of claims 1 to 14. The nucleic acid according to any one of claims 1 to 14 or the composition, kit or part of a kit according to any one of claims 15 to 19 for use as a medicine. The nucleic acid according to any one of claims 1 to 14 or the composition, kit or part of a kit according to any one of claims 15 to 19 for use in the treatment of cancer or tumor disease. A pharmaceutical composition comprising the nucleic acid according to any one of claims 1 to 14 or the composition according to any one of claims 15 to 18. The pharmaceutical composition according to claim 22, which comprises a pharmaceutically acceptable carrier. The nucleic acid according to any one of claims 1 to 14 or the composition, kit or part of a kit according to any one of claims 15 to 19 for increasing the expression of a encoded peptide or protein in vitro. use. a) A step of providing the nucleic acid according to any one of claims 1 to 14 or the composition according to any one of claims 15 to 18.
The b) the nucleic acid or the composition, a cell-free expression system, cell, or comprising a step of applying or administering to the organization, in vitro to increase the expression of the peptide or protein encoded Method.

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